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A strategy for augmenting multivalent glycoconjugates with a serum-protein recognition element extends half-life in vivo and maintains engagement with lectin receptors.

Since December 2019, the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread throughout the world. To eradicate it, it is crucial to acquire a strong and long-lasting anti-SARS-CoV-2 immunity, by either natural infection or vaccination. We collected blood samples 12–305 days after positive polymerase chain reactions (PCRs) from 35 recovered individuals infected by SARS-CoV-2. Peripheral blood mononuclear cells were stimulated with SARS-CoV-2-derived peptide pools, such as the spike (S), nucleocapsid (N) and membrane (M) proteins, and we quantified anti-S immunoglobulins in plasma. After 10 months post-infection, we observed a sustained SARS-CoV-2-specific CD4+ T-cell response directed against M-protein, but responses against S- or N-proteins were lost over time. Besides, we demonstrated that O-group individuals presented significantly lower frequencies of specific CD4+ T-cell responses against Pep-M than non O-group individuals. The non O-group subjects also needed longer to clear the virus, and they lost cellular immune responses over time, compared to the O-group individuals, who showed a persistent specific immune response against SARS-CoV-2. Therefore, the S-specific immune response was lost over time, and individual factors might determine the sustainability of the body’s defenses, which must be considered in the future design of vaccines to achieve continuous anti-SARS-CoV-2 immunity.

Synthetic glycoconjugates are used in the development of vaccines and the design of inhibitors for glycan-protein interactions. The

Atopic disorders are more common in children after heart transplant (HTx). We hypothesized that HTx at an early age and thymus excision (TE) affect development of T and B cells, especially regulatory T cells (Tregs), which help maintain tolerance.In this single-center study including 24 patients transplanted between 2013 and 2018, we investigated lymphocyte patterns in relation to these factors using flow cytometry. Clinical data were collected from standardized questionnaires and medical charts. Patients were stratified into TE and non-TE groups as well as patients with and without post-transplant atopy development/worsening.64% of TE patients experienced new or worsening asthma/eczema post-transplant compared to 20% of non-TE patients. TE patients had higher total Treg proportions (CD4+CD25+CD127lo) than non-TE patients (p = .043), but borderline significantly lower naïve Tregs (CD45RA+CD27-) (p = .057). Memory CD4+ T cells were higher in TE patients in trend (p = .084). Total Tregs did not differ between atopic/nonatopic groups, although naïve Tregs were significantly lower in atopic patients (p = .028). Memory CD4+ T cells were higher in atopic patients in trend (p = .082). IgM+IgD+ B cells were higher in nonatopic patients in trend (p = .064).New/worsening atopy is more common in thymectomized HTx children and is associated with alterations in T-cell profiles. Avoiding TE may prevent these alterations and reduce incidence of atopy post-HTx.

In chronic Hepatitis B Virus (HBV) infections HBV-specific T cells are functionally impaired. Immunotherapy may restore HBV-specific T cell responses essential for sustained disease remission off-treatment and induction of a functional cure. Chimigen® Molecules are fusion proteins of antigen(s) with the Fc fragment of a xenotypic antibody designed to target specific receptors on dendritic cells (DCs). Here we describe the production and pre-clinical evaluation of Chimigen® HBV (C-HBV), containing HBV PreS1 and PreS2 peptide fragments, HBV core and murine Fc, produced in insect cells. C-HBV binding to immature DCs and internalization by endocytosis was FcγRII (CD32) and mannose receptor (CD206) dependent and led to increased MHC I and MHC II surface expression. Upon exposure of human T cells isolated from HBV un-infected healthy and chronically HBV-infected donors to C-HBV-pulsed mature DCs ex vivo, C-HBV induced vigorous T cell proliferation and enhanced expression of IFN-γ, TNF-α, perforin and granzyme B in both CD4+ and CD8+ T cell subsets. Re-stimulation of C-HBV-activated T cells from chronically infected donors with HBV PreS1/PreS2 and core overlapping peptides induced IFN-γ production in both CD4+ and CD8+ populations. C-HBV-activation of peripheral blood mononuclear cells (PBMCs) from chronically HBV-infected patients stimulated granzyme B production by CD4+CD25− T responder (Tresp) cells, accompanied by an increase in Annexin V staining on CD4+CD25+ T regulatory (Treg) cell phenotype, consistent with apoptosis. The observed HBV-specific cellular responses induced by C-HBV ex vivo suggest that C-HBV is a promising immunotherapeutic candidate for the treatment of chronic HBV infections.

Since December 2019, the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread throughout the world. To eradicate it, it is crucial to acquire a strong and long-lasting anti-SARS-CoV-2 immunity, by either natural infection or vaccination. We collected blood samples 12–305 days after positive polymerase chain reactions (PCRs) from 35 recovered individuals infected by SARS-CoV-2. Peripheral blood mononuclear cells were stimulated with SARS-CoV-2-derived peptide pools, such as the Spike (S), Nucleocapsid (N), and Membrane (M) proteins, and we quantified anti-S immunoglobulins in plasma. After 10 months post-infection, we observed a sustained SARS-CoV-2-specific CD4+ T-cell response directed against M-protein, but responses against S- or N-proteins were lost over time. Besides, we demonstrated that A-group individuals presented significantly higher frequencies of specific CD4+ T-cell responses against Pep-M than O-group individuals. The A-group subjects also needed longer to clear the virus and they lost cellular immune responses over time, compared to the O-group individuals, who showed a persistent specific immune response against SARS-CoV-2. Therefore, the S-specific immune response was lost over time, and individual factors determine the sustainability of the body’s defences, which must be considered in the future design of vaccines to achieve continuous anti-SARS-CoV-2 immunity.SummaryThis work describes that cellular responses against SARS-CoV-2 M-protein can be detected after 10 months but were lost against S- and N-proteins. Moreover, the individual factors; ABO-group and age influence the sustainability of the specific humoral and cellular immunity against SARS-CoV-2.

Newborns are highly susceptible to infectious diseases. The underlying mechanism of neonatal infection susceptibility has generally been related to their under-developed immune system. Nevertheless, this notion has recently been challenged by the discovery of the physiological abundance of immunosuppressive erythroid precursors CD71+ erythroid cells (CECs) in newborn mice and human cord blood. Here, as proof of concept, we show that these cells are also abundant in the peripheral blood of human newborns. Although their frequency appears to be more variable compared to their counterparts in mice, they rapidly decline by 4 weeks of age. However, their proportion remains significantly higher in infants up to six months of age compared to older infants. We found CD45 expressing CECs, as erythroid progenitors, were the prominent source of reactive oxygen species (ROS) production in both humans and mice. Interestingly, a higher proportion of CD45+CECs was observed in the spleen versus bone marrow of neonatal mice, which was associated with a higher ROS production by splenic CECs compared to their siblings in the bone marrow. CECs from human newborns suppressed cytokine production by CD14 monocytes and T cells, which was partially abrogated by apocynin in vitro. Moreover, the depletion of CECs in neonatal mice increased the number of activated effector immune cells in their spleen and liver, which rendered them more resistant to Listeria monocytogenes infection. This was evident by a significant reduction in the bacteria load in the spleen, liver and brain of treated-mice compared to the control group, which enhanced their survival rate. Our finding highlights the immunoregulatory processes mediated by CECs in newborns. Thus, such tightly regulated immune system in newborns/infants may explain one potential mechanism for the asymptomatic or mild COVID-19 infection in this population.

Chimigen® HBV Immunotherapeutic Vaccine (C-HBV), a recombinant chimeric fusion protein comprising hepatitis B virus (HBV) S1 and S2 surface antigen fragments, Core antigen and a murine monoclonal antibody heavy chain fragment (Fc), was designed and produced in Sf9 insect cells. C-HBV targets the host immune system through specific receptors present on dendritic cells (DCs) which facilitates antigen internalization, processing, and presentation on MHC class I and II to induce both cellular and humoral immune responses against HBV antigens. T cell responses, previously assessed by ex vivo antigen presentation assays using human peripheral blood mononuclear cell (PBMC)-derived DCs and T cells from uninfected and HBV chronic-infected donors, demonstrated that C-HBV was highly immunogenic. A vaccine dose response study was performed in sheep to analyze the immunogenicity of C-HBV in vivo. Sheep (n = 8/group) received three consecutive subcutaneous injections of each dose of C-HBV at four-week intervals. Analysis of serum antibody levels confirmed C-HBV induced a dose-dependent antibody response to C-HBV and S1/S2-Core. Kinetics of the S1/S2-Core specific antibody response was similar to hepatitis B surface antigen (HBsAg)-specific antibody responses induced by ENGERIX-B. Analysis of cell-mediated immune responses (CMI) confirmed C-HBV induced both dose-dependent S1/S2-Core-specific lymphocyte proliferative responses and IFN-γ secretion. These responses were stronger with blood lymphocytes than with cells isolated from the lymph node draining the vaccination site. No correlation was seen between antibody titers and CMI. The results confirm C-HBV is an effective delivery vehicle for the induction of T cell responses and may be an appropriate candidate for immunotherapy for chronic HBV infections.

Purpose Accurate characterization of ABO-Ab is critical to assess impact in ABOi transplantation. The current ABO-Ab detection method using erythrocyte agglutination is limited by lack of ABO-subtype specificity, imprecise ABO-Ab isotype differentiation, and poor reproducibility. We previously developed an ABO-glycan microarray to address these limitations. Our aim here was to create a solid phase bead assay for ABO-Ab analysis. Methods ABO A- and B-subtype antigens (I,II,III,IV,V,VI) were coupled to Luminex beads and quantified using monoclonal ABO-Ab. Bovine serum albumin was coupled as a negative control bead. IgG and IgM isotypes with specificities for ABO A-subtypes were measured and compared in healthy adult plasma (n=28) by mean fluorescence intensity (MFI). These samples were also tested on the glycan array and by red cell agglutination. Results ABO-A and B-subtype-specific antibodies were detected, with high degree of variability in MFI values between subjects. IgG and IgM ABO-A and B-Ab were detectable in all non-AB subjects, although MFIs were low in some cases by both bead and array (Figure 1A). Group A and B subjects had similar IgM ABO-A-Ab levels to group O subjects, but lower IgG levels. The quantity of IgM isotype antibodies did not predict IgG level (Figure 1B). IgM Ab alone did not predict the RBC agglutination titre, as some subjects had predominantly IgG ABO-Ab. Conclusion Initial results of our Luminex ABO-Ab assay are promising for clinical laboratory use, where bead-based assays are already used for HLA-Ab. The specificity of this assay will allow precise assessment of ABO-Ab to antigen subtypes, known to be expressed differently in cardiac endothelium than erythrocytes (1). The ability to measure IgM and IgG ABO-Ab makes it possible to evaluate of the role of each in potential graft damage; isotype differentiation may be particularly relevant in the setting of plasmapheresis, which more efficiently removes IgM than IgG antibodies. 1. Jeyakanthan, M et al. AJT 2016; 16: 1548-1558

Purpose ABO histo-blood group incompatibility is a barrier in solid organ transplant due to the presence of ‘natural’ preformed ABO antibodies. However, ABO-incompatible (ABOi) heart transplantation is successful in infants as ABO antibodies are low/absent. A better understanding of the specificity and the production of ABO antibodies may allow for successful ABOi transplantation at older ages. Here, we sought to determine the isotype (IgM/IgG) and subtype (I-VI) specificity of ABO antibodies produced naturally or induced by sensitization in mice as a function of age and sex. Methods BALB/c mice were assessed for natural ABO antibody production over time (n=32/39, female/male; age 1-18 months), or challenged with human A erythrocytes (5 weekly intraperitoneal injection) beginning at 5 weeks of age to measure induced ABO antibody production (n=13/8, female/male; age 1-3 months). Plasma ABO antibody titre was determined by hemagglutination assay; ABO antibody isotype and subtype specificity were assessed by a novel ABH-glycan microarray created in our lab. Results Female mice produced markedly higher natural anti-A antibodies compared with male mice. With age, natural anti-A antibodies shifted from an IgM to IgG isotype in females but remained predominantly IgM in males (Figure 1). Most natural ABO antibodies were specific to antigen subtypes III/IV; specificity to subtypes I and II was absent or very low. In contrast, following A-antigen sensitization, both female and male mice produced comparable quantities of IgM and IgG anti-A antibodies with specificities for all subtypes (I-VI). Conclusion Male and female mice show previously undescribed differences in natural ABO antibody production. Markedly higher antibody production together with a distinct propensity to class switching suggest a more complex immune pathway in females than traditionally described for natural antibody to ‘T-independent’ carbohydrate antigens. Future studies will explore mechanisms for these sex differences and relevance to humans.

Background ABO-incompatible (ABOi) organ transplantation is performed owing to unremitting donor shortages. Defining mechanisms of antibody-mediated rejection, accommodation, and tolerance of ABOi grafts is limited by lack of a suitable animal model. We report generation and characterization of a murine model to enable study of immunobiology in the setting of ABOi transplantation. Methods Transgenesis of a construct containing human A1- and H-transferases under control of the ICAM-2 promoter was performed in C57BL/6 (B6) mice. A-transgenic (A-Tg) mice were assessed for A-antigen expression by histology and flow cytometry. B6 wild-type (WT) mice were sensitized with blood group A-human erythrocytes; others received passive anti-A monoclonal antibody and complement after heart transplant. Serum anti-A antibodies were assessed by hemagglutination. "A-into-O" transplantation (major histocompatibility complex syngeneic) was modeled by transplanting hearts from A-Tg mice into sensitized or nonsensitized WT mice. Antibody-mediated rejection was assessed by morphology/immunohistochemistry. Results A-Tg mice expressed A-antigen on vascular endothelium and other cells including erythrocytes. Antibody-mediated rejection was evident in 15/17 A-Tg grafts in sensitized WT recipients (median titer, 1:512), with 2 showing hyperacute rejection and rapid cessation of graft pulsation. Hyperacute rejection was observed in 8/8 A-Tg grafts after passive transfer of anti-A antibody and complement into nonsensitized recipients. Antibody-mediated rejection was not observed in A-Tg grafts transplanted into nonsensitized mice. Conclusions A-Tg heart grafts transplanted into WT mice with abundant anti-A antibody manifests characteristic features of antibody-mediated rejection. These findings demonstrate an effective murine model to facilitate study of immunologic features of ABOi transplantation and to improve potential diagnostic and therapeutic strategies.

Purpose ABO-incompatible organ transplants (ABOi-Tx) are at high-risk of rapid rejection mediated by preformed ABO antibodies (Ab), except during infancy when these are absent. ABO Abs are produced naturally in humans and mice without obvious exposure to ABH antigens (Ag) by mechanisms that remain poorly understood, although early studies suggested a role for bacteria in stimulation of ABO Ab production. Our group showed T cells are needed to induce anti-A Ab following immunization of wild-type (WT) mice with human blood group A (Hu A-RBC), however it's unclear whether natural ABO Ab production is also T cell-dependent. Furthermore, sex is an important biological factor in immune responses and may play a role in ABO production. Herein, we investigated the roles of sex, microbiome, and T cells for the production of natural ABO Ab. Methods Male and female WT, CD4, MHC-II, and T cell knock-out (KO) mice (B6 background, 6-7 weeks old) received either i.p. injections of Hu A-RBC (weekly x3) or were untreated. Serum anti-A Ab was measured by hemagglutination using RBCs. To study the role of gut bacteria in ABO Ab production, either drinking water was supplemented with antibiotics, or sera were collected from germ-free mice. Results Table: WT of both sexes produced increasing amounts of natural anti-A with age. In the KO mice, females produced significantly higher natural ABO Ab than males or WT females. Hu A-RBC injection did not induce anti-A Ab in the KO mice. Antibiotics significantly reduced natural ABO Ab production in CD4KO mice. Surprisingly, germ-free female mice produced natural ABO Ab without exposure to ABO-Ag. Conclusion In contrast to induced Ab, natural ABO Ab develops in the absence of CD4 T cells (T-dependent). The production of dramatically increased natural anti-A Ab by KO females over males suggests a sex-linked role for CD4 T cells in regulation of natural ABO Ab production. Unlike the common paradigm, bacteria are not absolutely required for natural ABO Ab production.

Purpose ABO-incompatible heart transplantation (ABOi HTx) is safe in young children and increases donor access. Post-ABOi HTx, B cell tolerance develops to donor ABO blood group antigen(s) by mechanisms not fully defined. To study ABO tolerance, we developed A-transgenic mice (A-Tg) that express A-antigen on vascular endothelium, erythrocytes (RBCs) and lymphocytes; we demonstrated A-antigen-specific tolerance induced by HTx into 4 wk-old, MHC-identical, wild-type (WT) mice. Intentional induction of tolerance may allow subsequent ABOi HTx. Recently, we showed ABO tolerance could be intentionally induced in WT mice after ip injection of infant mice with intact A-Tg blood cells (BCs). Herein, we sought to determine specific A-Tg cell type(s) capable of inducing tolerance. Methods WT BALB/c (BALB) mice at 7 days of age were left untreated or injected ip (weekly × 3) with either unfractionated BCs, erythrocytes (RBCs), peripheral blood mononuclear cells (PBMCs), or splenocytes from A-Tg BALB mice (see Table). Two weeks later, all mice were injected ip (weekly × 5) with human A-RBC (A-antigen challenge) in an attempt to elicit anti-A antibody (Ab) production. Serum anti-A and third-party (non-A anti-human) Ab were assessed by hemagglutination or ABH glycan microarray. Results In response to challenge with A-antigen, high levels of anti-A Ab were produced both in untreated mice and in mice previously treated with A-Tg PBMCs or splenocytes (Table). In contrast, anti-A Ab remained undetectable/very low in mice treated as infants with A-Tg BCs or RBCs. Third-party Ab responses were high for all groups. Conclusion A-Tg RBCs induced robust A-antigen-specific tolerance in infant mice, whereas A-Tg lymphocytes (PBMCs, splenocytes) did not. Future studies will explore mechanisms of A-Tg RBC tolerance induction with the goal to design synthetic ABH-multivalent polyethylene glycol (PEG) glycoconjugates for intentional ABO tolerance to allow subsequent ABOi HTx safely.

Aim ABO antibody (ABO-Ab) titration is performed for ABO-incompatible (ABOi) transplantation management and is traditionally assessed by hemagglutination. However, hemagglutination cannot readily differentiate Ab isotype, nor distinguish between Abs to ABO-subtype antigens, which are expressed differently on organs vs erythrocytes. Our goal was to explore Luminex as a potential platform for the detection of IgG and IgM antibodies to ABO subtype antigens. Methods Phase I: Human blood group antigen subtype A-II was synthesized and conjugated to bovine serum albumin (BSA). BSA-A-II conjugates were coupled to Luminex beads. Coupling was assessed using phycoerythrin (PE)-labeled mouse monoclonal IgG and IgM anti-human ABO-A. Increasing BSA-A-II antigen concentrations were tested to optimize MFI. A Luminex-200 was used for bead acquisition. Phase II: Antigens ABO A-III and A-IV were similarly tested (alone and multiplexed) using additional Luminex beads. Phase III: Dilutions of PE-labeled anti-human IgG conjugate were tested against dilutions of anti-ABO-A plasma (blood group B donor) in a checkerboard titration. BSA-A-II coupled beads were used. Results Bead coupling was optimal at 5ug of BSA-A-II antigen. The coupling of BSA-A-II, -III and -IV was successful as demonstrated by a linear increase in MFI with increasing concentration of primary antibody (Figure). Beads were successfully labeled with monoclonal ABO-Ab when multiplexed. The assay appears most sensitive with the lowest background when patient plasma is diluted 1/25. Conclusions HLA laboratories are uniquely situated to assess ABO antibodies by Luminex as expertise and instrumentation already exist. This novel assay has the potential to be adopted by clinical HLA laboratories for rapid, specific, and sensitive assessment of IgM/IgG ABO subtype-specific ABO antibodies in ABOi transplantation. We continue to optimize the assay, include additional controls, and assess additional ABO subtypes. We are also testing flow-based bead platforms. Download : Download high-res image (185KB) Download : Download full-size image

Aim The frequency and impact of AT1R antibodies in pediatric heart transplantation has not been well studied. Our aim was to measure AT1R antibodies (AT1R-Ab) in this patient population and pediatric controls and to assess for non-specific reactivity in this assay. Methods We included 42 patients ( n = 154 samples) for whom pre- and post-transplant sera and HLA Ab data were available. AT1R-Ab were measured by ELISA (One Lambda Inc.). Samples were tested/interpreted as per product insert. Age-matched, sex-balanced, non-transplant patient controls ( n = 27) were collected from cardiac–catheterization laboratory. A subset with positive results ( n = 52 sera from 20 patients) was re-tested following Adsorb Out (One Lambda Inc.). Analysis was performed using GraphPad. Results No significant difference was observed between patient and control AT1R antibody levels (Fig. 1). Values over 40 U/ml were detected in 38% of patient sera. Adsorbed sera had significantly decreased AT1R values vs non-adsorbed sera (Fig. 2A). There was no significant change in AT1R-Ab from pre- to post-transplant overall in either group but a higher proportion of patients changed from positive to negative using adsorbed vs non-adsorbed sera (20% vs 12.5%) (Fig. 2B and C). Conclusions A high proportion of patients and controls were positive for AT1R-Ab, however, many sera appear to be false positive, possibly due to BSA reactivity or incomplete blocking in the assay. As more assays for non-HLA Ab are added into our clinical algorithms, appropriate controls must be included. A ‘blank’ well would be useful in this assay for accurate interpretation of results. Association of the adsorbed and non-adsorbed AT1R-Ab to transplant outcomes is currently being assessed.

Aim Accurate, clinically relevant characterization of ABO antibodies (ABO-Ab) is critical to assess their impact in ABO incompatible transplantation. The current ABO-Ab detection method using erythrocyte agglutination is limited by lack of ABO subtype specificity, difficulty in ABO-Ab isotype differentiation, and poor reproducibility. We previously developed a slide micro-array method for ABO-Ab analysis to address these limitations. Our aim was to create a similar bead solid phase assay. Methods ABO A subtype antigens (I, II, III, IV, V, VI) were coupled to Luminex beads and quantified using monoclonal ABO-Ab. Bovine serum albumin and alpha-Gal antigen were coupled as negative/positive control beads, respectively. Optimal plasma and anti-human IgG and IgM labelling-antibody dilutions were determined. IgG and IgM isotypes with specificities for ABO A-subtypes were measured (n = 40 healthy donors) by mean fluorescent intensity (MFI). Levels of IgG vsIgM ABO-A-Ab were compared (Wilcoxon signed-rank test). Preliminary positive MFI thresholds for each ABO A-subtype bead were calculated based on ABO-A-Ab in ABO A control plasma. Results Variation in ABO-Ab levels between ABO A-subtypes was detected. There was no significant difference between IgG and IgM ABO-Ab levels in paired MFI for all ABO types. IgG and IgM ABO-Ab are clearly detectable in all non-ABO A controls. Calculated MFI thresholds accurately interpreted positive ABO-A-Ab in ABO O samples for the IgG assay and most beads in the IgM assay. ABO B controls had similar IgM ABO-A-Ab levels to ABO O controls but lower levels of IgG. Reactivity to alpha-Gal was present but inconsistent. Conclusions This bead-based method successfully measures ABO-A-Ab and shows promise for clinical laboratory implementation. The specificity of these solid-phase assays will facilitate accurate assessment of ABO-Ab to subtypes, which are known to be expressed differently in endothelium than on erythrocytes. The ability to accurately measure both IgM and IgG ABO antibodies makes it possible to evaluate of the role of each ABO-Ab isotype in transplantation; this may be particularly relevant in the setting of plasmapheresis, which more efficiently removes IgM antibodies. Further optimization of this assay, controls, and ABO B and ABO H panel development are underway.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4, CD152) is an inhibitory T cell receptor predominately expressed on activated T cells. The duck CTLA-4 (DuCTLA-4) cDNA and a transcript lacking the predicted transmembrane encoding region (DuCTLA-4DeltaTM) were isolated from splenocytes using RT-PCR. The predicted DuCTLA-4 protein showed an identity of 92%, 49% and 47% with chicken, human and mouse homologues, respectively. Sequence comparison revealed conservation of residues implicated in the B7 ligand binding, disulfide linkages, glycosylation and intracellular signaling. DuCTLA-4 mRNA was predominately expressed in primary and secondary immune organs. DuCTLA-4 and DuCTLA-4DeltaTM transcripts were differentially regulated in PBMCs. Flow cytometric analysis showed constitutive expression of DuCTLA-4 protein on freshly isolated PBMCs and a modest increase upon mitogen stimulation. Our observations suggest that DuCTLA-4 and its isoform DuCTLA-4DeltaTM evolved before the divergence of birds and mammals. Both DuCTLA-4 isoforms have significant structural homology to mammalian CTLA-4 proteins but their individual roles in the regulation of duck immune responses remains to be elucidated.

Cytotoxic T lymphocytes and natural killer cells destroy target cells via the directed exocytosis of lytic effector molecules such as perforin and granzymes. The mechanism by which these proteins enter targets is uncertain. There is ongoing debate over whether the most important endocytic mechanism is nonspecific or is dependent on the cation-independent mannose 6-phosphate receptor. This study tested whether granzyme B endocytosis is facilitated by dynamin, a key factor in many endocytic pathways. Uptake of and killing by the purified granzyme B molecule occurred by both dynamin-dependent and -independent mechanisms. However most importantly, serglycin-bound granzyme B in high-molecular-weight degranulate material from cytotoxic T lymphocytes predominantly followed a dynamin-dependent pathway to kill target cells. Similarly, killing by live cytotoxic T lymphocytes was attenuated by a defect in the dynamin endocytic pathway, and in particular, the pathways characteristically activated by granzyme B were affected. We therefore propose a model where degranulated serglycin-bound granzymes require dynamin for uptake.

The serine proteinase granzyme B is crucial for the rapid induction of target cell apoptosis by cytotoxic T cells. Granzyme B was recently demonstrated to enter cells in a perforin-independent manner, thus predicting the existence of a cell surface receptor(s). We now present evidence that this receptor is the cation-independent mannose 6-phosphate/insulin-like growth factor receptor (CI-MPR). Inhibition of the granzyme B–CI-MPR interaction prevented granzyme B cell surface binding, uptake, and the induction of apoptosis. Significantly, expression of the CI-MPR was essential for cytotoxic T cell-mediated apoptosis of target cells in vitro and for the rejection of allogeneic cells in vivo. These results suggest a novel target for immunotherapy and a potential mechanism used by tumors for immune evasion.

In TCR-αβ transgenic mice, CD4−CD8− TCR-αβ+ (αβ DN) cells arise in the absence of positively selecting MHC molecules and are resistant to clonal deletion in Ag-expressing mice. In this study the activation requirements and functional properties of αβ double-negative (DN) cells were compared with those of positively selected CD8+ cells expressing equivalent levels of the same MHC class I-restricted transgenic TCR. We found that positively selected CD8+ cells required a lower density of the antigenic ligand for optimal proliferative responses compared with αβ DN cells derived from nonpositively selecting mice. However, when the CD8 coreceptor on CD8+ cells was blocked with an anti-CD8 mAb, both αβ DN and CD8+ cells exhibited the same dose-response curve to the antigenic ligand and the same dependence on CD28/B7 costimulation. Positively selected CD8+ cells also differed from αβ DN cells in that they differentiated into more efficient killers and IL-2 producers after Ag stimulation, even after CD8 blockade. However, Ag-activated αβ DN and CD8+ cells were equally efficient in producing IFN-γ, suggesting that this functional property is independent of positive selection. We also found that αβ DN cells recovered from the lymph nodes of Ag-expressing mice were functionally anergic. This anergic state was associated with defective proliferation and IL-2 production in response to Ag stimulation. These observations indicate that αβ DN cells can be anergized in vivo by physiological levels of the antigenic ligand.

The issue of whether the signaling process during positive selection can affect the efficiency by which the positively selected T cells respond to antigenic stimulation has not been addressed. We approached this question by determining the consequences of positive selection of a particular transgenic TCR (2C TCR) in the H-2b and the H-2k thymus. The H-2b thymus provides a strong positive-selecting environment for the 2C TCR, whereas the H-2k thymus selects weakly for the 2C TCR. Although the positively selected CD8 thymocytes from the H-2b or H-2k thymus expressed similar levels of the CD8 coreceptor molecule, those for the H-2k thymus expressed a slightly lower level of the 2C TCR. This lower level of 2C TCR expression by H-2k CD8 thymocytes was not a result of coexpression of endogenous TCRs. Interestingly, CD8 thymocytes from H-2k mice were hyporesponsive to Ag stimulation compared with those from the H-2b mice. The functional maturity of positively selected CD8 thymocytes from the H-2b or H-2k thymus was inversely correlated with the level of heat stable Ag expressed by these cells. Furthermore, TCR-derived signals appear to be more efficiently coupled to downstream pathways leading to proliferation and cytokine production in CD8 thymocytes from H-2b 2C mice than those derived from H-2k 2C mice. These results provide the first demonstration that the intensity of the signaling process during positive selection affects the efficiency by which TCR-derived signals in positively selected thymocytes are coupled to downstream effector pathways.

The affinity/avidity model for T cell development postulates that ligands with high affinity for the TCR are efficient in negative selection, whereas those with lower affinity/avidity favor positive selection. Using the 2C TCR transgenic model, we evaluated the efficacy of ligands with widely differing affinity for the TCR (3 × 103 to 2 × 106 M−1) in mediating thymocyte deletion. The relative affinities of the 2C TCR for the p2Ca/Ld, dEV-8/Kb, p2Ca-A3/Ld, and p2Ca/Kb ligands are approximately 1000:50:10:1, respectively. Here we show, using an in vitro assay, that the deletion of 2C CD4+CD8+ thymocytes is mediated not only by p2Ca/Ld, but also by the lower affinity ligands dEV-8/Kb, p2Ca-A3/Ld, and p2Ca/Kb, albeit at relatively higher peptide concentrations. Deletion mediated by low affinity ligands required CD8, whereas high affinity ligand-mediated deletion was CD8 independent. The p2Ca/Kb and dEV-8/Kb ligands are naturally occurring in H-2b mice, and others have shown that p2Ca/Kb can induce the maturation of CD4−CD8+2C-TCRhigh thymocytes in fetal thymic organ culture. In this study we showed that in addition to deletion, the p2Ca/Kb and dEV-8/Kb ligands, in the presence of exogenous IL-2, induced mature 2C T cell proliferation, albeit at a lower level than that induced by the high affinity p2Ca/Ld ligand. Thus, the same low affinity ligands that can effect negative selection and, in the case of p2Ca/Kb, the maturation of CD8 single-positive thymocytes can also induce the activation of mature CD8 T cells.

The ileal Peyer's patch (PP) in sheep plays a central role in the development and production of B cells. Associated with a tremendous amount of B cell proliferation in this site is the extensive diversification of the Ig repertoire by somatic hypermutation. Very few (< 5%) of the B cells produced in the ileal PP differentiate and emigrate; instead, the vast majority of these cells soon die, and we have previously shown that death is associated with apoptosis. When placed in culture, ileal PP B cells die rapidly by apoptosis, such that after 24 h, 60 +/- 1% of DNA is fragmented. Here, we show that the extent of this spontaneous B cell apoptosis in culture, as quantitated by DNA fragmentation, was significantly increased in a dose-dependent manner by the glucocorticoids hydrocortisone or dexamethasone. Furthermore, treatment of lambs with 2-2.5 mg/kg of dexamethasone resulted in a marked increase in the number of apoptotic cells in the ileal PP and an increase in ileal PP B cell DNA fragmentation to 20 +/- 6%, compared with 2.4 +/- 0.1% in untreated lambs. Anti-immunoglobulin (Ig) antibodies also increased the extent of DNA fragmentation in cultured ileal PP B cells. After 24 or 48 h of culture with anti-Ig (PIg47A), DNA fragmentation was 74 +/- 2% and 75 +/- 3%, respectively. Ileal PP B cells are rescued from apoptosis by agents that activate protein kinase C and increase cytosolic Ca2+, and here we show that this treatment also results in apoptotic rescue in the presence of dexamethasone or anti-Ig. We speculate that the apoptosis of ileal PP B cells in situ may be modulated by glucocorticoids and by the cross-linking of surface Ig. Apoptosis, induced by a signal through surface Ig, may be an important mechanism in the deletion of self-reactive B cells during the expansion of the Ig repertoire in the ileal PP.

The ileal Peyer's patch (PP) is the major site of B cell production and is a site of immunoglobulin gene diversification in the sheep. Within the ileal PP follicles there is both intense proliferation and death of B cells. We have previously demonstrated that most, if not all of this death can be attributed to apoptosis. Likewise, ileal PP B cells die rapidly by apoptosis in culture--after 6 h many cells appear pyknotic and about 50% of cellular DNA is fragmented. We now show that the DNA fragmentation and cell death of ileal PP B cells can be almost completely abrogated during the first 12 h of culture by the addition of the phorbol esters, phorbol dibutyrate (PBu2) or phorbol myristate acetate. This inhibition of apoptosis could be sustained for greater than 24 h by the concomitant addition of both PBu2 and the Ca2+ ionophore A23187. However, the rescue of B cells from apoptosis by PBu2, with or without Ca2+ ionophore, was prevented by macromolecular synthesis inhibitors or inhibitors of protein kinase C activation. Furthermore, treatment of cultures with PBu2, with or without Ca2+ ionophore, resulted in an activated B cell phenotype and a three- to fourfold increase in cell proliferation. We conclude that protein kinase C activation in conjunction with an increase in intracellular [Ca2+] can provide the signals necessary to rescue ileal PP B cells from apoptosis, and speculate that these ileal PP B cells are destined to die unless they receive a signal that rescues them from the death pathway.

The ABO histo-blood group system is the most important antigen system in transplantation medicine, yet no small animal model of the ABO system exists. To determine the feasibility of developing a murine model, we previously subcloned the human alpha-1,2-fucosyltransferase (H-transferase, EC 2.4.1.69) cDNA and the human alpha-1,3-N-acetylgalactosaminyltransferase (A-transferase, EC 2.4.1.40) cDNA into lentiviral vectors to study their ability to induce human histo-blood group A antigen expression on mouse cells. Herein we investigated the optimal conditions for human A and H antigen expression in murine cells. We determined that transduction of a bicistronic lentiviral vector (LvEF1-AH-trs) resulted in the expression of A antigen in a mouse endothelial cell line. We also studied the in vivo utility of this vector to induce human A antigen expression in mouse liver. After intrahepatic injection of LvEF1-AH-trs, A antigen expression was observed on hepatocytes as detected by immunohistochemistry and real-time RT-PCR. In human group A erythrocyte-sensitized mice, A antigen expression in the liver was associated with tissue damage, and deposition of antibody and complement. These results suggest that this gene transfer strategy can be used to simulate the human ABO blood group system in a murine model. This model will facilitate progress in the development of interventions for ABO-incompatible transplantation and transfusion scenarios, which are difficult to develop in clinical or large animal settings.

The ileal Peyer's patch (PP) and the bursa of Fabricius have major roles in populating the B cell system in sheep and chickens, respectively. These tissues contain greater than 90% B cells and possess a massive proliferation index with greater than 5% of B cells entering mitosis per hour. Paradoxically, almost all of the B cells produced in these sites rapidly die in situ. Here we show that the extensive B cell death occurring in the ileal PP and bursa is associated with apoptosis. Gel electrophoresis of ileal PP cell DNA from 7-14 week-old lambs and bursal cell DNA from 4-week-old chickens demonstrated a laddering of DNA in multiples of approximately 200 bp, a pattern indicative of apoptosis. In sheep, the intensity of the laddering pattern seen after agarose gel electrophoresis was always greater with ileal PP cell DNA compared with thymocyte DNA, and usually greater than jejunal PP cell DNA. Likewise, DNA isolated from chicken bursal cells and mouse PP cells always exhibited a more intense laddering pattern than chicken or mouse thymocytes, respectively. When placed in culture ileal PP cells died rapidly less than 40% viable cells were recovered after 24 h. Within 6 h of culture many ileal PP cells exhibited an apoptotic appearance in that they contained condensed chromatin and fragmented nuclei. Moreover, greater than 55% of total cellular DNA was fragmented. Compared with thymocytes, ileal PP cells underwent DNA fragmentation to a much greater extent and with a faster time course in short-term culture. We propose that cell death by apoptosis may make an important contribution to B cell development in the lamb ileal PP and the chicken bursa. Apoptosis may provide a mechanism for the diversification of the B cell immune repertoire and/or the selection of non-self reactive B cells.