SIRPγ-CD47 Interaction Positively Regulates the Activation of Human T Cells in Situation of Chronic Stimulation.

A numerous number of positive and negative signals via various molecules modulate T-cell activation. Within the various transmembrane proteins, SIRPγ is of interest since it is not expressed in rodents. SIRPγ interaction with CD47 is reevaluated in this study. Indeed, we show that the anti-SIRPγ mAb clone LSB2.20 previously used by others has not been appropriately characterized. We reveal that the anti-SIRPα clone KWAR23 is a Pan anti-SIRP mAb which efficiently blocks SIRPα and SIRPγ interactions with CD47. We show that SIRPγ expression on T cells varies with their differentiation and while being expressed on Tregs, is not implicated in their suppressive functions. SIRPγ spatial reorganization at the immune synapse is independent of its interaction with CD47. In vitro SIRPα-γ/CD47 blockade with KWAR23 impairs IFN-γ secretion by chronically activated T cells. In vivo in a xeno-GvHD model in NSG mice, the SIRPγ/CD47 blockade with the KWAR23 significantly delays the onset of the xeno-GvHD and deeply impairs human chimerism. In conclusion, we have shown that T-cell interaction with CD47 is of importance notably in chronic stimulation.

Versatile and rapid microfluidics-assisted antibody discovery.

Recent years have seen unparalleled development of microfluidic applications for antibody discovery in both academic and pharmaceutical research. Microfluidics can support native chain-paired library generation as well as direct screening of antibody secreting cells obtained by rodent immunization or from the human peripheral blood. While broad diversities of neutralizing antibodies against infectious diseases such as HIV, Ebola, or COVID-19 have been identified from convalescent individuals, microfluidics can expedite therapeutic antibody discovery for cancer or immunological disease indications. In this study, a commercially available microfluidic device, Cyto-Mine, was used for the rapid identification of natively paired antibodies from rodents or human donors screened for specific binding to recombinant antigens, for direct screening with cells expressing the target of interest, and, to our knowledge for the first time, for direct broad functional IgG antibody screening in droplets. The process time from cell preparation to confirmed recombinant antibodies was four weeks. Application of this or similar microfluidic devices and methodologies can accelerate and enhance pharmaceutical antibody hit discovery.

Phosphatidylserine-Targeting Monoclonal Antibodies Exhibit Distinct Biochemical and Cellular Effects on Anti-CD3/CD28-Stimulated T Cell IFN-γ and TNF-α Production.

Phosphatidylserine (PS)-targeting monoclonal Abs (mAbs) that directly target PS and target PS via ß2-gp1 (ß2GP1) have been in preclinical and clinical development for over 10 y for the treatment of infectious diseases and cancer. Although the intended targets of PS-binding mAbs have traditionally included pathogens as well as stressed tumor cells and its associated vasculature in oncology, the effects of PS-targeting mAbs on activated immune cells, notably T cells, which externalize PS upon Ag stimulation, is not well understood. Using human T cells from healthy donor PBMCs activated with an anti-CD3 + anti-CD28 Ab mixture (anti-CD3/CD28) as a model for TCR-mediated PS externalization and T cell stimulation, we investigated effects of two different PS-targeting mAbs, 11.31 and bavituximab (Bavi), on TCR activation and TCR-mediated cytokine production in an ex vivo paradigm. Although 11.31 and Bavi bind selectivity to anti-CD3/28 activated T cells in a PS-dependent manner, surprisingly, they display distinct functional activities in their effect on IFN-γ and TNF-ɑ production, whereby 11.31, but not Bavi, suppressed cytokine production. This inhibitory effect on anti-CD3/28 activated T cells was observed on both CD4+ and CD8+ cells and independently of monocytes, suggesting the effects of 11.31 were directly mediated by binding to externalized PS on activated T cells. Imaging showed 11.31 and Bavi bind at distinct focal depots on the cell membrane. Collectively, our findings indicate that PS-targeting mAb 11.31 suppresses cytokine production by anti-CD3/28 activated T cells.

Nanovesicles released by OKT3 hybridoma express fully active antibodies.

Recent findings have shown that nanovesicles preparations from either primary immune cells culture supernatants or plasma contain immunoglobulins, suggesting that a natural way of antibody production may be through exosome release. To verify this hypothesis, we used the OKT3 hybridoma clone, which produces a murine IgG2a monoclonal antibody used to reduce rejection in patients undergoing organ transplantation. We showed exosome-associated immunoglobulins in hybridoma supernatants, by Western blot, nanoscale flow cytometry and immunocapture-based ELISA. The OKT3-exo was also being able to trigger cytokines production in both CD4 and CD8 T cells. These results show that nanovesicles contain immunoglobulin and could be used for immunotherapy. These data could lead to a new approach to improve the effectiveness of therapeutic antibodies by exploiting their natural property to be expressed on nanovesicle membrane, that probably render them more stable and as a consequence more capable to interact with their specific ligand in the best way.

Developing an Arrayed CRISPR-Cas9 Co-Culture Screen for Immuno-Oncology Target ID.

Immunotherapies including PD-L1 blockade have shown remarkable increases in the T cell-directed antitumor response; however, efficacy is seen only in a minority of patients. Recently, pooled CRISPR-Cas9 knockout (CRISPRn) screens in tumor/immune co-culture systems have identified a number of genes that confer resistance to T cell killing in pathways including antigen presentation and cytokine signaling, providing insight into tumor mechanisms that cause resistance to immunotherapies. The development of an arrayed CRISPRn screen in a tumor/immune co-culture system would allow the identification of novel targets for immuno-oncology, characterization of hits from pooled screens, and multiple assay endpoints to be measured per gene. Here, a small-scale arrayed CRISPRn screen was successfully developed to investigate the effects on a co-culture of T cells and Cas9-expressing PC9 lung adenocarcinoma cells modified to express anti-CD3 antibody on the cell surface (PC9-OKT3 T cell system). A focused CRISPRn library was designed to target genes involved in known resistance mechanisms (including antigen presentation, cytokine signaling, and apoptosis) as well as genes involved in immune synapse interactions. The viability of PC9 cells was assessed in two-dimensional adherent co-cultures via longitudinal imaging analysis. Knockout of epidermal growth factor receptor (EGFR) and PLK1 in tumor cells cultured alone or with T cells resulted in increased tumor cell death, as expected, whereas knockout of the test gene ICAM1 showed subtle donor-specific resistance to T cell killing. Taken together, these data provide proof of concept for arrayed CRISPRn screens in tumor/immune co-culture systems and warrant further investigation of in vitro co-culture models.

Resident T Cells in Resolved Psoriasis Steer Tissue Responses that Stratify Clinical Outcome.

Psoriasis is driven by focal disruptions of the immune-homeostasis in human skin. Local relapse following cessation of therapy is common and unpredictable, which complicates clinical management of psoriasis. We have previously shown that pathogenic resident T cells accumulate in active and resolved psoriasis, but whether these cells drive psoriasiform tissue reactions is less clear. Here, we activated T cells within skin explants using the pan-T cell activating antibody OKT-3. To explore if T cells induced different tissue response patterns in healthy and psoriasis afflicted skin, transcriptomic analyses were performed with RNA-sequencing and Nanostring. Core tissue responses dominated by IFN-induced pathways were triggered regardless of the inflammatory status of the skin. In contrast, pathways induced by IL-17A, including Defensin beta 2 and keratinocyte differentiation markers, were activated in psoriasis samples. An integrated analysis of IL-17A and IFN-related responses revealed that IL-17 dominated tissue response correlated with early relapse following UVB treatment. Stratification of tissue responses to T cell activation in resolved lesions could potentially offer individualized prediction of disease relapse during long-term immunomodulatory treatment.

Novel T cells with improved in vivo anti-tumor activity generated by RNA electroporation.

The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB). The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy.

Severity of the TGN1412 trial disaster cytokine storm correlated with IL-2 release.

AIM: To determine if cytokine release with a solid phase assay is predictive of adverse responses for a range of therapeutic mAbs. METHODS: Cytokine ELISAs and a multi-array system were used to compare responses generated by different therapeutic mAbs using a solid phase assay. Flow cytometry was employed to determine the cellular source of those cytokines. RESULTS: Only TGN1412 and muromonab-CD3 stimulated CD4+ T-cell mediated cytokine release characterized by significant (all P < 0.0001) IFNγ, TNFα, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17 and IL-22 release, comparable with T-cell mitogen. Significantly greater (P < 0.0001) IL-2 release with TGN1412 (2894-6051 pg ml⁻¹) compared with muromonab-CD3 (62-262 pg ml⁻¹) differentiated otherwise comparable cytokine responses. Likewise, TGN1412 stimulated significantly more (P = 0.0001) IL-2 producing CD4+ T-cells than muromonab-CD3 and induced Th1, Th2, Th17 and Th22 subsets that co-release this cytokine. Significant TNFα release was observed with bevacizumab (P = 0.0001), trastuzumab (P = 0.0031) and alemtuzumab (P = 0.0177), but no significant IL-2 release. TGN1412 and muromonab-CD3 caused pro-inflammatory cytokine release despite significantly (both P < 0.0001) increasing numbers of T-cells with a regulatory phenotype. CONCLUSIONS: The severity of the adverse response to TGN1412 compared with muromonab-CD3 and other therapeutic mAbs correlates with the level of IL-2 release.

Characterization of the cellular immunity in patients presenting extensive dermatophytoses due to Trichophyton rubrum.

Dermatophytes invade the stratum corneum of the skin and other keratinized tissues such as hair and nails, and Trichophyton rubrum causes approximately 80% of cutaneous mycoses in humans. To evaluate the cellular immune response of patients with extensive dermatophytosis caused by T. rubrum, we evaluated lymphocyte populations, the lymphoproliferative response to: phytohaemagglutinin (PHA); anti-CD3 (OKT3); and pokeweed mitogen (PWM), Candida sp. (CMA), an extract of T. rubrum, and the main fungal epitope TriR2 (T). We also evaluated interleukin (IL)-4, IL-10, IL-12 and IFN-γ after stimulation by PHA, CMA and TriR2. The immunophenotyping showed no differences between patients and controls. The lymphoproliferation test showed significant differences between the groups stimulated by PWM and CMA, as well as against TriR2, being significantly higher for the control group. Conversely, there were similar results for the groups after stimulation by the extract. The cytokines' quantification showed a significant difference between the groups only for IFN-γ stimulated by PHA and TriR2. We can conclude that the fungal extract can stimulate lymphoproliferation by both groups' lymphocytes. However, the response to Tri r2 was more specific. We showed that some patients with extensive dermatophytosis have normal cellular response, recognising both the extract and TriR2.

Upregulated TCRζ enhances interleukin-2 production in T-cells from patients with CML.

T-cell immunodeficiency is a common feature in patients with chronic myeloid leukemia (CML), and deficiency in CD3 levels was detected in T cells from these patients, which may represent a characteristic that is related to a lower T cell activation. In this study, we explored the possibility that forced TCRζ gene expression may upreg-u-late T cell receptor (TCR) signaling activation and reverse interleukin-2 (IL-2) production in T cells from patients with CML. A recombinant eukaryotic vector expressing TCRζ was transfected into T cells by nucleofection. Phosphorylated TCRζ, phosphorylated NF-κB, and the IL-2 level in TCRζ-transfected CD3+T cells that were activated with anti-CD3 and anti-CD28 antibodies were measured by Western blot and enzyme-linked immunosorbent assay (ELISA). Significantly increased TCRζ levels were found in TCRζ-transfected CD3+T cells. After CD3 and CD28 antibody stimulation, a significantly higher phosphorylated TCRζ chain level was demonstrated, and an increased IL-2 production in TCRζ-upregulated T cells was associated with the increased expression of the phosphorylated NF-κB. In conclusion, TCRζ gene transfection could restore TCRζ chain deficiency and enhance IL-2 production in T cells from patients with CML. It is possible that TCRζ chain reconstitution in leukemia-specific, clonally expanded T cells will effectively increase their activation of antileukemia cytotoxicity.

A stepwise protocol to coat aAPC beads prevents out-competition of anti-CD3 mAb and consequent experimental artefacts.

Artificial antigen-presenting cells (aAPC) are widely used for both clinical and basic research applications, as cell-based or bead-based scaffolds, combining immune synapse components of interest. Adequate and controlled preparation of aAPCs is crucial for subsequent immunoassays. We reveal that certain proteins such as activatory anti-CD3 antibody can be out-competed by other proteins (e.g. inhibitory receptor ligands such as PDL1:Fc) during the coating of aAPC beads, under the usually performed coating procedures. This may be misleading, as we found that decreased CD8 T cell activity was not due to inhibitory receptor triggering but rather because of unexpectedly low anti-CD3 antibody density on the beads upon co-incubation with inhibitory receptor ligands. We propose an optimized protocol, and emphasize the need to quality-control the coating of proteins on aAPC beads prior to their use in immunoassays.

A humanised mouse model of cytokine release: comparison of CD3-specific antibody fragments.

CD3-specific antibodies have shown clinical efficacy in both transplantation and autoimmunity. However, targeting CD3 in this way can lead to T-cell activation and a serious cytokine release syndrome mediated by Fcγ receptor binding. An in vivo mouse model has been developed using severe combined immunodeficient (SCID) mice to detect human T-cell depletion and cytokine release into the circulation after administration of OKT3. This system has been used to evaluate OKT3 antibody fragments lacking the entire Fc region alongside whole antibody constructs. These data clearly show that cytokine release is detected with all OKT3 antibody constructs and fragments tested and these can be ranked from highest to lowest as follows: mIgG2a>hIgG1 (Ala-Ala)>hIgG1 diFab' maleimide (DFM)>hIgG1 F(ab')2>mIgG2a F(ab')2>hIgG1 Fab'. Furthermore, the monovalent hIgG1 Fab' fragment gives the least cytokine release but it does not deplete human T-cells in this assay format. This suggests that T-cell activation may be playing a role in the mechanism of action of anti-CD3 antibodies and consequently the unwanted cytokine release is potentially unavoidable for this class of molecules. This model system provides a useful tool to aid in understanding and reducing the potential risks of cytokine release following antibody therapy.

A proteomic view at T cell costimulation.

The "two-signal paradigm" in T cell activation predicts that the cooperation of "signal 1," provided by the T cell receptor (TCR) through engagement of major histocompatility complex (MHC)-presented peptide, with "signal 2″ provided by costimulatory molecules, the prototype of which is CD28, is required to induce T cell effector functions. While the individual signalling pathways are well understood, little is known about global changes in the proteome pattern during TCR/CD28-mediated activation. Therefore, comparative 2-DE-based proteome analyses of CD3(+) CD69(-) resting T cells versus cells incubated with (i) the agonistic anti-CD3 antibody OKT3 mimicking signal 1 in absence or presence of IL-2 and/or with (ii) the agonistic antibody 15E8 triggering CD28-mediated signaling were performed. Differentially regulated spots were defined leading to the identification of proteins involved in the regulation of the metabolism, shaping and maintenance of the cytoskeleton and signal transduction. Representative members of the differentially expressed protein families, such as calmodulin (CALM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), L-lactate dehydrogenase (LDH), Rho GDP-dissociation inhibitor 2 (GDIR2), and platelet basic protein (CXCL7), were independently verified by flow cytometry. Data provide a detailed map of individual protein alterations at the global proteome level in response to TCR/CD28-mediated T cell activation.

Tumor PD-L1 co-stimulates primary human CD8(+) cytotoxic T cells modified to express a PD1:CD28 chimeric receptor.

Tumors exploit immunoregulatory checkpoints that serve to attenuate T cell responses as a means of circumventing immunologic rejection. Programmed death ligand 1 (PD-L1) is a negative regulator of T cell function and is frequently expressed by solid tumors. By engaging programmed death 1 (PD-1) on activated T cells, PD-L1(+) tumors directly render tumor-specific T cells, including adoptively transferred T cells, functionally exhausted. As a strategy to overcome tumor PD-L1 effects on adoptively transferred T cells, we sought to convert PD-1 to a T cell costimulatory receptor by exchanging its transmembrane and cytoplasmic tail with that of CD28. Rather than becoming exhausted upon engagement of PD-L1(+) tumors, we hypothesized that CD8(+) cytotoxic T lymphocytes (CTL) genetically modified to express this PD1:CD28 chimera would exhibit enhanced functional attributes. Here we show that cell surface expressed PD1:CD28 retains the capacity to bind PD-L1 resulting in T cell costimulation as evidenced by increased levels of ERK phosphorylation, augmentation of cytokine secretion, increased proliferative capacity, and enhanced expression of effector molecule Granzyme B. We provide evidence that this chimera could serve as a novel engineering strategy to overcome PD-L1 mediated immunosuppression.

Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help.

BACKGROUND: Using in vivo mouse models, the mechanisms of CD4+ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4+ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4+ T cell help of CD8+ T cell proliferation using a novel in vitro model. METHODS/PRINCIPAL FINDINGS: We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3+ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4+ T cells was associated with significantly improved CD8+ T cell expansion in vitro. The CD4+ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4+ T cell help of CD8+ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8+ T cells. CONCLUSIONS: We have developed an in vitro model that advances our understanding of the immunobiology of human CD4+ T cell help of CD8+ T cells. Our data suggests that human CD4+ T cell help can be leveraged to expand CD8+ T cells in vitro.

Activated monocytes prime naïve T cells against autologous cancer: vigorous cancer destruction in vitro and in vivo.

It has not been considered so far that antigen-presenting cells (APC) may phagocytose immunogenic material from autologous cancer cells. Assuming the presence of cancer-immunogenic material in APC, we developed a novel autologous priming method that does not require tumour cells or identified peptides. Cancer-immunogenic information came from CD14+ monocytes. When stimulated with CD3-activated T cells, monocytes primed CD3+ CD4+ and CD3+ CD8+ resting/naïve T cells to become powerful effector cells within 24 h. During priming, depletion of CD14+ monocytes but not CD1a+ CD83+ dendritic cells prevented T cell priming. During cancer cell destruction, dendritic cells, but not monocytes, enhanced cancer cell lysis. The cascade-primed (CAPRI) immune cell quartet comprising monocytes, dendritic cells, CD4+ T and CD8+ T cells induced a significant decrease in the number of suppressive CD25(high) FoxP3+ CD4+ T cells. CAPRI cells induced a marked upregulation of MHC class I and class II expression in cancer cells, which is crucial for autoimmune-like lysis. We show in vivo evidence of the CAPRI cell concept in nude mice. In humans, we present circumstantial clinical evidence showing the efficacy of CAPRI cells in an adjuvant treatment attempt for breast cancer patients with metastasis (N = 42). Compared to patients at the Munich Tumor Center (no CAPRI treatment N = 428), almost double the expected number of patients survived 5 years (P =1.36 × 10⁻¹4). The CAPRI method is a safe procedure without negative side effects. High numbers of cancer-specific CAPRI cells can be obtained within a week against different cancer types for efficient adoptive cell therapy.

Preculture of PBMCs at high cell density increases sensitivity of T-cell responses, revealing cytokine release by CD28 superagonist TGN1412.

Human volunteers receiving TGN1412, a humanized CD28-specific monoclonal antibody, experienced a life-threatening cytokine release syndrome during a recent trial. Preclinical tests using human PBMCs had failed to announce the rapid release of TNF, IFN-γ, and other toxic cytokines in response to this CD28 "superagonist" (CD28SA). CD28SA activate T-lymphocytes by ligating CD28 without overt engagement of the TCR. They do, however, depend on "tonic" TCR signals, which they amplify. Here we show that short-term preculture of PBMCs at high, but not at low, cell density results in massive cytokine release during subsequent stimulation with soluble TGN1412. Restoration of reactivity was cell-contact dependent, involved functional maturation of both monocytes and T cells, was sensitive to blockade by HLA-specific mAb, and was associated with TCR polarization and tyrosine phosphorylation. CD4 effector memory T cells were identified as the main source of proinflammatory cytokines. Importantly, responses to other T-cell activating agents, including microbial antigens, were also enhanced if PBMCs were first allowed to interact under tissue-like conditions. We provide a protocol, which strongly improves reactivity of circulating T cells to soluble stimulants, thereby allowing for more reliable preclinical testing of both activating and inhibitory immunomodulatory drugs.

Teplizumab for treatment of type 1 diabetes (Protégé study): 1-year results from a randomised, placebo-controlled trial.

BACKGROUND: Findings of small studies have suggested that short treatments with anti-CD3 monoclonal antibodies that are mutated to reduce Fc receptor binding preserve ß-cell function and decrease insulin needs in patients with recent-onset type 1 diabetes. In this phase 3 trial, we assessed the safety and efficacy of one such antibody, teplizumab. METHODS: In this 2-year trial, patients aged 8-35 years who had been diagnosed with type 1 diabetes for 12 weeks or fewer were enrolled and treated at 83 clinical centres in North America, Europe, Israel, and India. Participants were allocated (2:1:1:1 ratio) by an interactive telephone system, according to computer-generated block randomisation, to receive one of three regimens of teplizumab infusions (14-day full dose, 14-day low dose, or 6-day full dose) or placebo at baseline and at 26 weeks. The Protégé study is still underway, and patients and study staff remain masked through to study closure. The primary composite outcome was the percentage of patients with insulin use of less than 0·5 U/kg per day and glycated haemoglobin A(1c) (HbA(1C)) of less than 6·5% at 1 year. Analyses included all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, number NCT00385697. FINDINGS: 763 patients were screened, of whom 516 were randomised to receive 14-day full-dose teplizumab (n=209), 14-day low-dose teplizumab (n=102), 6-day full-dose teplizumab (n=106), or placebo (n=99). Two patients in the 14-day full-dose group and one patient in the placebo group did not start treatment, so 513 patients were eligible for efficacy analyses. The primary outcome did not differ between groups at 1 year: 19·8% (41/207) in the 14-day full-dose group; 13·7% (14/102) in the 14-day low-dose group; 20·8% (22/106) in the 6-day full-dose group; and 20·4% (20/98) in the placebo group. 5% (19/415) of patients in the teplizumab groups were not taking insulin at 1 year, compared with no patients in the placebo group at 1 year (p=0·03). Across the four study groups, similar proportions of patients had adverse events (414/417 [99%] in the teplizumab groups vs 98/99 [99%] in the placebo group) and serious adverse events (42/417 [10%] vs 9/99 [9%]). The most common clinical adverse event in the teplizumab groups was rash (220/417 [53%] vs 20/99 [20%] in the placebo group). INTERPRETATION: Findings of exploratory analyses suggest that future studies of immunotherapeutic intervention with teplizumab might have increased success in prevention of a decline in ß-cell function (measured by C-peptide) and provision of glycaemic control at reduced doses of insulin if they target patients early after diagnosis of diabetes and children. FUNDING: MacroGenics, the Juvenile Diabetes Research Foundation, and Eli Lilly.