Primate testing of TGN1412: right target, wrong cell.

The failure of toxicity studies in non-human primates to predict the cytokine release syndrome during a first-in-man study of the CD28-specific monoclonal antibody TGN1412 has remained unexplained so far. In this issue of the BJP, work from the NIBSC first identifies the effector memory subset of human T-lymphocytes as the most likely source of the pro-inflammatory cytokines released during the study, and goes on to show that in cynomolgus monkeys, this subset lacks CD28, the target molecule of TGN1412. We discuss the implications for the TGN1412 catastrophe and for preclinical evaluation of biologicals in animal models in general.

Superagonistic CD28 antibody induces donor-specific tolerance in rat renal allografts.

The ultimate goal of organ transplantation is to establish graft tolerance where CD4+CD25+FOXP3+ regulatory T (Treg) cells play an important role. We examined whether a superagonistic monoclonal antibody specific for CD28 (CD28 SA), which expands Treg cells in vivo, would prevent acute rejection and induce tolerance using our established rat acute renal allograft model (Wistar to Lewis). In the untreated or mouse IgG-treated recipients, graft function significantly deteriorated with marked destruction of renal tissue, and all rats died by 13 days with severe azotemia. In contrast, 90% of recipients treated with CD28 SA survived over 100 days, and 70% survived with well-preserved graft function until graft recovery at 180 days. Analysis by flow cytometry and immunohistochemistry demonstrated that CD28 SA induced marked infiltration of FOXP3+ Treg cells into the allografts. Furthermore, these long-surviving recipients showed donor-specific tolerance, accepting secondary (donor-matched) Wistar cardiac allografts, but acutely rejecting third-party BN allografts. We further demonstrated that adoptive transfer of CD4+CD25+ Treg cells, purified from CD28 SA-treated Lewis rats, significantly prolonged allograft survival and succeeded in inducing donor-specific tolerance. In conclusion, CD28 SA treatment successfully induces donor-specific tolerance with the involvement of Treg cells, and thus the therapeutic value of this approach warrants further investigation and preclinical studies.

Combination of donor-specific blood transfusion with anti-CD28 antibody synergizes to prolong graft survival in rat liver transplantation.

Donor-specific blood transfusion (DST) has been shown to effectively induce tolerance to certain allografts. In addition, it is well known that blockade of costimulatory signals reduces the ability of T cells to respond to alloantigens, prolonging allograft survival in some transplant models. We assessed the effects of single or multiple DSTs in the absence or presence of anti-CD28 monoclonal antibodies (mAbs) on graft function and host survival in rat liver transplantation (LTx). Fully MHC-mismatched adult male Dark Agouti (DA) and Lewis (LEW) rats were used as donors and recipients, respectively. Heparinized DA blood was administered to naïve LEW rats 7 days before LTx [DST(-7d)], 14 and 7 days before LTx [DST(1 x 2)], twice a week for 2 weeks prior to LTx [DST(2 x 2)] and once a week for 4 weeks prior to LTx [DST(1 x 4)]. For some experiments, two different monoclonal antibodies (mAb) to rat CD28 (JJ316 and JJ319) were administered in combination with some DST treatments. We found that DST administration induced a time- and dose-dependent increase in host survival. Treatment of LEW rats with JJ316 or JJ319 mAb alone failed to prolong graft survival over untreated rats; however, the combination of DST(1 x 2) with JJ316 or JJ319 mAb induced indefinite survival at 100 days following surgery. We found that this protective effect was associated with increased numbers of splenic CD4+ CD45RC- but not CD4+ CD25+ foxp3+ T-cells in long-term survivors. Our data suggest that the combination of suboptimal DST with CD28 mAb induces donor-specific tolerance that correlates with enhanced numbers of regulatory T-cells.

Costimulation and autoimmune diabetes in BB rats.

Costimulatory signals regulate T-cell activation. To investigate the role of costimulation in autoimmunity and transplantation, we studied the BB rat model of type 1 diabetes. Diabetes-prone BB (BBDP) rats spontaneously develop disease when 55-120 days of age. We observed that two anti-CD28 monoclonal antibodies (mAb) with different functional activities completely prevented diabetes in BBDP rats. Anti-CD154 mAb delayed diabetes, whereas treatment with CTLA4-Ig or anti-CD80 mAb accelerated disease. Anti-CD86 or anti-CD134L mAbs had no effect. Diabetes resistant BB (BBDR) rats are disease-free, but >95% of them develop diabetes after treatment with polyinosinic-polycytidylic acid and an mAb that depletes Treg cells. In the induced BBDR model, anti-CD154 mAb delayed onset of diabetes, whereas CTLA4-Ig, anti-CD134L or either of the anti-CD28 mAbs had little or no effect. In contrast, blockade of the CD134-CD134L pathway was highly effective for preventing autoimmune recurrence against syngeneic islet grafts in diabetic BBDR hosts. Blockade of the CD40-CD154 pathway was also effective, but less so. These data suggest that the effectiveness of costimulation blockade in the treatment of type 1 diabetes is dependent on both the costimulatory pathway targeted and the mechanism of induction, stage, intensity and duration of the pathogenic process.

Superagonistic anti-CD28 antibodies: potent activators of regulatory T cells for the therapy of autoimmune diseases.

This paper reviews the existing evidence regarding the use of superagonistic anti-CD28 antibodies (CD28 superagonists) for therapeutic manipulation of regulatory T cells (T(reg) cells). The molecular properties of superagonistic anti-CD28 antibodies allow the generation of a strong activating signal in mature T cells, including T(reg) cells, without additional stimulation of the T cell receptor complex. CD28 superagonist administration in vivo leads to the preferential expansion and strong activation of naturally occurring CD4+CD25+CTLA-4+FoxP3+ T(reg) cells over conventional T cells. In animal models, both prophylactic and therapeutic administration of a CD28 superagonist prevented or at least greatly mitigated clinical symptoms and induced remission. Adoptive transfer experiments have further shown that CD28 superagonists mediate protection by expansion and activation of CD4+CD25+ T(reg) cells. Therefore, superagonistic anti-CD28 antibodies offer a promising novel treatment option for human autoimmune diseases and the first clinical trials are eagerly awaited.

Molecular and cellular mechanisms of T cell development.

The thymus is central to the establishment of a functioning immune system. Here is the place where T cells mature from hematopoietic progenitors, driven by mutual interactions of stromal cells and the developing thymocytes. As a result, different types of T cells are generated, all of which have been carefully selected for the ability to act in host defense towards non-self and against the potential to mount pathogenic self-reactive autoimmune responses. In this review we summarize our present knowledge on the lineage decisions taking place during this development, the selection processes responsible for shaping the T cell antigen-receptor repertoire, the interactions with the stromal components and the signal transduction pathways which transform the interactions with the thymic microenvironment into cellular responses of survival, proliferation, differentiation and, importantly, also of cell death.

HIV type 1 abrogates TAP-mediated transport of antigenic peptides presented by MHC class I. Transporter associated with antigen presentation.

Downregulation of MHC class I expression following human immunodeficiency virus 1 (HIV-1) infection is thought to play an important role in viral escape from immune recognition by cytotoxic T-lymphocytes (CTLs). Since exogenous addition of HIV-1-derived peptides restores susceptibility of HIV-1-infected cells to CTL-mediated lysis, we tested whether endogenous peptide loading is impaired in these cells. Our results show that in HIV-1-infected cells the ability of the transporter associated with antigen presentation (TAP) to translocate antigenic peptides from the cytosol to the lumen of the ER for presentation on MHC class I molecules is abolished. These data suggest that interference with the supply of antigenic peptides to the MHC class I pathway provides an additional mechanism by which HIV-1 evades the CTL-mediated immune response.

Control of T cell hyperactivation in IL-2-deficient mice by CD4(+)CD25(-) and CD4(+)CD25(+) T cells: evidence for two distinct regulatory mechanisms.

In IL-2-deficient mice, antigen-activated CD4 T cells accumulate and cause lethal immune pathology. Wild-type cells of hematopoietic origin present in the same animal are able to prevent this hyperactivation of T cells, but the mechanisms and cells controlling the IL-2-deficient cells are unknown. Here we show that IL-2(-) CD4 cells with an ovalbumin-specific transgenic TCR (IL-2(-) OVAtg) undergo both clonal expansion and clonal contraction when transferred to euthymic recipients and challenged with antigen, but continuously expand in athymic hosts. Cotransfer of wild-type CD4 T cells prevents the accumulation of IL-2-deficient cells. On the residual IL-2(-) TCRtg cells CD69 and CD25 are up-regulated, suggesting that activation per se is not suppressed and that the cells had received an IL-2 signal. Since IL-2 is able to restore the defective antigen-induced cell death (AICD) of IL-2-deficient T cells in vitro, paracrine IL-2 provided by the wild-type CD4 cells may thus be able to allow clonal contraction of IL-2-deficient cells also in vivo. Interestingly however, regulatory CD4(+)CD25(+) cells also efficiently contain the clone size of antigen-stimulated IL-2-deficient T cells. Since CD4(+)CD25(+) cells do not produce IL-2, this suggests a mechanism of suppression distinct from paracrine IL-2 delivery. In keeping with this, the residual IL-2(-) TCRtg cells recovered after cotransfer of regulatory CD4(+)CD25(+) cells do not show increased CD25 or CD69 expression, suggesting that they had not received paracrine IL-2 and that clonal containment occurred at the level of initial activation rather than clonal contraction by AICD. IL-2 deficiency therefore may upset T cell homeostasis by two distinct mechanisms: the failure to program expanding T cells for apoptosis, and the failure to generate functional CD4(+)CD25(+) regulatory cells.

Genetic control of peripheral TCRAV usage by representation in the preselection repertoire and MHC allele-specific overselection.

TCRAV segments contribute significantly to MHC restriction as illustrated by their general preference for either the CD4 or CD8 T cell subset and additional, MHC allele-specific overselection during T cell differentiation. The 10-fold over-representation of the TCRAV8S2 (VA8S2) segment in CD8 over CD4 T cells by the RT1(f) haplotype of LEW.1F rats provides the most striking example of MHC allele-specific overselection of a VA segment reported so far. Also in alloreactivity, VA8S2(+) CD8 cells from RT1(f-) rats are preferentially expanded by RT1(f+) stimulators. We have identified the class I molecule, A(f), mediating VA8S2 overselection and report that it differs only in four amino acids at the MHC-TCR interface from the class I molecule A(a), which is neutral with regard to selection of VA8S2. We also provide an extensive survey of the TCRAV8 family and show that among 14 functional VA8 segments in LEW rats, the dramatic A(f)-dependent overselection is unique for VA8S2. Surprisingly, VA8S2 expression in CD8 T cells of RT1(f+) rats derived from a Sprague-Dawley stock was only 3% as compared to the 12% observed in LEW.1F. The VA8S2 segment of Sprague-Dawley (VA8S2(SD)) differs from VA8S2 of the LEW background (VA8S2(l)) in only two amino acids, one of which is located in CDR2 and could thus participate in allele-specific recognition of A(f). However, analysis of the pre- and postselection thymic repertoires of Sprague-Dawley and LEW.1F rats and of the repertoire of CD8 cells from both strains expanded in the alloreactive response to RT1(f) revealed that the difference in VA8S2 representation between the two backgrounds is explained by differential availability in the preselection repertoires and not by a difference in overselection. Sequence comparisons of A(f) and A(a) and of both VA8S2 segments suggest a predominant role of CDR1 in hyper-reactivity to A(f). Thus, the VA composition of the mature TCR repertoire is influenced by TCRA: locus polymorphisms at two levels: the regulation of VA usage in the preselection repertoire and the composition of structural elements which contribute to specific VA-MHC interactions during thymic selection.

Thymic development and repertoire selection: the rat perspective.

This review summarizes our current knowledge of T-cell maturation and repertoire selection in the rat thymus. Some unique features of early thymocyte development and of CD4/CD8 lineage decision are described. A detailed analysis of lineage progression through the CD4, CD8 "double positive" compartment and T-cell receptor-induced CD8 T-cell maturation in cell culture is provided. A second emphasis is placed on interactions between germline-encoded T-cell receptor elements with MHC molecules in thymic repertoire selection and alloreactivity

Autonomous induction of proliferation, JNK and NF-alphaB activation in primary resting T cells by mobilized CD28.

Induction of proliferation in primary resting T cells requires engagement of both the antigen-specific TCR and the co-stimulatory receptor CD28. Here we report that CD28 functions as an autonomous mitogenic receptor which is mobilized by TCR signaling through cytoskeletal rearrangement. Shortcutting of TCR-dependent CD28 recruitment by stimulation with monoclonal antibodies specific for mobilized CD28 results in maximum proliferation and IL-2 secretion in primary resting T cells without activation of ZAP-70, a central component of the TCR's signal transduction machinery. Engagement of mobilized CD28 fully activates the c-Jun N-terminal kinase cascade and translocation of NF-alphaB, two key targets of signal integration in co-stimulation. We propose a two-step activation model for co-stimulation in primary resting T cells in which antigen recognition recruits co-stimulatory receptors which then autonomously transduce signals promoting T cell proliferation.

MEK activity regulates negative selection of immature CD4+CD8+ thymocytes.

CD4+CD8+ thymocytes are either positively selected and subsequently mature to CD4 single positive (SP) or CD8 SP T cells, or they die by apoptosis due to neglect or negative selection. This clonal selection is essential for establishing a functional self-restricted T cell repertoire. Intracellular signals through the three known mitogen-activated protein (MAP) kinase pathways have been shown to selectively guide positive or negative selection. Whereas the c-Jun N-terminal kinase and p38 MAP kinase regulate negative selection of thymocytes, the extracellular signal-regulated kinase (ERK) pathway is required for positive selection and T cell lineage commitment. In this paper, we show that the MAP/ERK kinase (MEK)-ERK pathway is also involved in negative selection. Thymocytes from newborn TCR transgenic mice were cultured with TCR/CD3epsilon-specific Abs or TCR-specific agonist peptides to induce negative selection. In the presence of the MEK-specific pharmacological inhibitors PD98059 or UO126, cell recovery was enhanced and deletion of DP thymocytes was drastically reduced. Furthermore, development of CD4 SP T cells was blocked, but differentiation of mature CD8 SP T cells proceeded in the presence of agonist peptides when MEK activity was blocked. Thus, our data indicate that the outcome between positively and negatively selecting signals is critically dependent on MEK activity.

Expression of T-cell receptor beta-chain mRNA and protein in gamma/delta T-cells from euthymic and athymic rats: implications for T-cell lineage divergence.

The relationship between alpha/beta and gamma/delta T-cell lineages was studied in rats using RT-PCR analysis of TCRbeta transcripts in gamma/delta T-cell hybridomas and an intracellular staining technique to detect TCRbeta protein in primary gamma/delta T-cells. We report the presence of functional TCRbeta transcripts in 2/9 gamma/delta T-cell hybridomas. About 15% of peripheral gamma/delta T-cells and thymocytes also express TCRbeta protein, giving a minimum estimate for successful Tcrb rearrangement based on ex vivo single cell analysis. In athymic rats, gamma/delta T-cells expressing intracellular beta protein are present but at a lower frequency than in euthymic controls, suggesting that in the thymus, more gamma/delta T-cell precursors pass through a stage where functional beta rearrangement has occurred than in extrathymic sites. Analysis of TCR expression in purified transitory immature CD4-8+ (iCD8SP) thymocytes and their spontaneously developing CD4+8+ (DP) progeny showed that TCRy mRNA is expressed in iCD8SP cells but not in their immediate DP progeny that reinitiate RAG-I transcription and commence alpha/betaTCR expression. We conclude that rat gamma/delta T cells can separate from the alpha/beta lineage after TCRbeta expression, but not after entry into the DP compartment.

Triggering of T cell proliferation through CD28 induces GATA-3 and promotes T helper type 2 differentiation in vitro and in vivo.

The relative contribution of T cell receptor-versus CD28-mediated signals in co-stimulation of resting CD4 T cells is thought to influence their functional differentiation towards T helper (Th) 1 versus Th2 subsets. We have used a conventional and a mitogenic CD28-specific monoclonal antibody to assess the effect of polyclonal T cell activation through CD28 alone on CD4 subset differentiation. In vivo, mitogenic but not conventional anti-CD28 induces massive lymphocytosis, the Th2 cytokines interleukin (IL)-4 and IL-10, and Th2-dependent immunoglobulin isotypes, most notably IgE. In vitro, it is shown that mitogenic anti-CD28 primes for IL-4-dependent induction of IL-4 expression much more efficiently than conventional co-stimulation. At the molecular level, we show for the first time that the activation of the "Th2 promoting" transcription factor GATA-3 requires co-stimulation by CD28 and is also induced by mitogenic anti-CD28 alone. We suggest that CD28-dependent induction of GATA-3 in concert with other transcription factors, which are preferentially induced by strong CD28-signals, primes CD4 T cells for IL-4-dependent Th2 differentiaton.

Ligation of the CD4 receptor induces activation-independent down-regulation of L-selectin.

Lymphocyte circulation plays an important role in the generation of a specific immune response. Mature lymphocytes continuously circulate between blood and lymph, entering the lymphoid tissue via high endothelial venules. Trafficking across high endothelial venules of peripheral lymph nodes (PLN) depends on the expression of L-selectin. It has been shown that L-selectin is rapidly cleaved from the surface by a metalloproteinase after in vitro activation. Here, we show that ligation of CD4, without ligation of the T cell receptor for antigen, causes down-regulation of L-selectin on T helper cells. This down-regulation is caused by proteolytic cleavage by a metalloproteinase and is reversible by the addition of hydroxamic acid-based metalloproteinase inhibitors. We show that in vivo down-regulation of L-selectin in huCD4tg mice by mAb reduces the homing of lymphocytes to PLN in adoptive transfer experiments. Because CD4 is a coreceptor for HIV-1, the down-regulation of L-selectin induced by CD4 ligation could play a role in the pathogenesis of AIDS. We provide evidence that CD4 ligation by HIV-1 induces metalloproteinase-dependent L-selectin down-regulation. Reduced levels of L-selectin expression might contribute to immune deficiency in individuals infected with HIV by inhibiting T cell redistribution and decreasing the probability of an encounter between specific lymphocytes and viral antigens in PLN.

Impaired thymopoietic potential of immature CD3(-)CD4(+)CD8(-) T cell precursors from SIV-infected rhesus monkeys.

Immature thymocyte subpopulations were examined for their capacity to differentiate in a newly developed xenogeneic monkey-mouse fetal thymus organ culture (FTOC) system. We provide evidence for impaired precursor function of CD3(-)CD4(+)CD8(-) thymocytes after in vivo infection with SIVmac251 as indicated by a reduced cell number per FTOC and a lower percentage of thymocytes with more mature phenotypes. Addition of recombinant SIV glycoprotein 120 (rgp120) also resulted in a dose-dependent impairment of T cell maturation in FTOC. The data suggest that in patients infected with HIV, T cell maturation and thus replenishment of peripheral pools may be compromised as a result of intrathymic infection or circulating viral gp120.

Differential CD4/CD8 subset-specific expression of highly homologous rat Tcrb-V8 family members suggests a role of CDR2 and/or CDR4 (HV4) in MHC class-specific thymic selection.

Different rat Tcrb haplotypes express either TCR beta variable segment (Tcrb-V) 8.2l or 8.4a. Both V segments bind the mAb R78 but differ by one conservative substitution (L14V) and clusters of two and four substitutions in the complementarity-determining region (CDR) 2 and CDR4 [hypervariable loop 4 (HV4)]. Independently of MHC alleles numbers of R78+ CD4+ cells are lower in Tcrb-V8.2l-expressing than in Tcrb-V8.4a-expressing strains. Expression of R78+ TCR during T cell development, analysis of backcross populations and generation of a Tcrb congenic strain [LEW.TCRB(AS)] define two mechanisms how Tcrb haplotypes affect the frequency of R78+ cells, one acting prior to thymic selection leading to up to 2-fold higher frequency of Tcrb-V8.4a versus Tcrb-V8.2l in unselected thymocytes and another occurring between the TCRlow and the CD4/CD8 single-positive stage. The latter leads to a 50% reduction of frequency of Tcrb-V8.4a CD8+ cells but not CD4+ cells and does not affect either subset of Tcrb-V8.2l cells. A comparison of rat classical class I MHC (RT1.A) sequences and current models of TCR-MHC-peptide interaction suggests that this reduction in frequency of Tcrb-V8.4a CD8 cells may be a consequence of differential selection of Tcrb-V8.2l versus Tcrb-V8.4a TCR by differential binding of CDR2beta to highly conserved areas of C-terminal parts of the alpha helices of class I MHC molecules.

Prolonged allograft survival but no tolerance induction by modulating CD28 antibody JJ319 after high-responder rat heart transplantation.

BACKGROUND: Allograft rejection depends on T cell immune responses requiring antigen recognition and costimulatory signals through accessory T cell receptors, including CD28. Inhibition of CD28 signaling with a CTLA-4-immunoglobulin (Ig) fusion protein has resulted in immunosuppression and occasional T cell anergy in mouse transplant models, but not in rats. Because this approach also inhibits a potentially tolerizing signal through CTLA-4, selective blockade of CD28 ligation might induce more profound immunosuppression and transplant tolerance. METHODS: The effects of escalating doses of the rat CD28 monoclonal antibody JJ319 on allograft survival were studied after vascularized heterotopic heart transplantation in a high responder strain combination (DA to Lewis). CD28 antigen modulation and circulating antibody levels were monitored by flow cytometry. RESULTS: CD28 antibody JJ319 markedly prolonged cardiac graft survival compared with untreated controls (7 days, range: 6-8). A strictly dose-dependent increase in median graft survival time was demonstrated with a maximum of 36 days (range: 30-40; p <0.001) after the administration of 8 x 1 mg JJ319 i.p. (days -1 to +6 before/after transplantation). However, indefinite graft survival and tolerance could not be induced by JJ319 treatment. At the maximal dose, flow cytometry showed complete down modulation of the CD28 receptor for 10-14 days without T cell depletion in close temporal relation to antibody presence in serum. In vitro, CD28-modulated T cells showed significantly reduced responses to activation. CONCLUSIONS: CD28 antibody JJ319 induces profound immunosuppression after rat heart transplantation, however without development of transplant tolerance. The underlying mechanism seems to be receptor modulation during primary alloantigen recognition. While still potentially applicable clinically, there are no qualitative or quantitative differences to the treatment with CTLA-4/lg or the blockade of CD2 or LFA-1, as reported elsewhere. Thus, a CD28-modulating approach seems not to allow therapeutic exploitation of a tolerizing signal delivered by CTLA-4 but may still be clinically applicable, especially in combined immune interventions.

In vitro skewing of TCR transgenic CD4+ T cells from interleukin-2 deficient mice towards Th1 and Th2 in the absence of exogenous interleukin-2.

Previous experiments from several groups have indicated that in vitro priming for Th2 cells rigorously requires IL-4 but also depends on IL-2 [1-3]. On the other hand, IL-2 deficient mice characteristically have highly increased serum levels of the Th2-dependent isotypes IgG1 and IgE [4]. The overproduction of these isotypes is lost in IL-2 x IL-4 double deficient animals [5]. To readdress the question of a need for IL-2 for Th2 skewing in vitro we used T cells from IL-2-/- mice also transgenic for the DO11.10 TCR which is specific for OVA + IAd [6]. CD4+ cells from these mice were primed in vitro on IL-2-/- dendritic cells in the presence of OVA peptide and IL-4, IL-12 and IL-15, respectively. Following restimulation, cytokine production was analysed by intracellular staining with anti IL-4 and anti IFNgamma antibodies and flow cytometry. The data show that IL-4 primes IL-2-/- T cells for IL-4 production even in the absence of exogenous IL-2, while IL-12, as expected, polarises towards IFNgamma production. The ability to be primed for IL-4 production in the absence of IL-2 was also exhibited by naive CD4+CD62LlowTCR transgenic IL-2-/- cells and thus was not restricted to the CD44high CD62Llow cells which make up a high proportion of CD4+ cells in IL-2 deficient mice. We conclude that IL-2 is not absolutely required for in vitro skewing of naive T cells towards Th2.