Short-term treatment with CD3 antibody preserves residual beta-cell function for at least 18 months in patients with recent-onset type 1 diabetes.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

CD3-specific antibodies have the unique capacity to restore self-tolerance in established autoimmunity. They induce long-term remission of overt diabetes in nonobese diabetic (NOD) mice and in human type I diabetes. The underlying mechanisms had been unclear until now. Here we report that treatment with CD3epsilon-specific antibodies induces transferable T-cell-mediated tolerance involving CD4+CD25+ cells. However, these CD4+CD25+ T cells are distinct from naturally occurring regulatory T cells that control physiological autoreactivity. CD3-specific antibody treatment induced remission in NOD Cd28-/- mice that were devoid of such regulatory cells. Remission of diabetes was abrogated by coadministration of a neutralizing transforming growth factor (TGF)-beta-specific antibody. The central role of TGF-beta was further suggested by its increased, long-lasting production by CD4+ T cells from tolerant mice. These data explain the intriguing tolerogenic effect of CD3-specific antibodies and position them as the first clinically applicable pharmacological stimulant of TGF-beta-producing regulatory CD4+ T cells.

Anti-CD3 monoclonal antibodies suppress immune responses by transient T-cell depletion and antigenic modulation of the CD3/T-cell receptor complex. Anti-CD3 treatment of adult nonobese diabetic (NOD) mice, a spontaneous model of T-cell-mediated autoimmune insulin-dependent diabetes mellitus, significantly inhibits the autoimmune process. Short-term low-dose anti-CD3 treatment (5 ,ug/day i.v. for 5 consecutive days) prevented the occurrence of an accelerated form of the disease induced by cyclophosphamide. More unexpectedly, when applied to adult NOD females within 7 days of the onset of full-blown diabetes, the same anti-CD3 regimen induced a complete remission of overt disease (i.e., a return to permanent normoglycemia) in 64-80% of mice. This remission was durable (>4 months) and was not associated with the disappearance of insulitis (mononuclear cell infiltration of the islets). The immunosuppression was apparently specific for fl-cell-associated antigens, since mice showing anti-CD3-induced remission rejected histoincompatible skin grafts normally, whereas they did not destroy syngeneic islet grafts, unlike control untreated overtly diabetic NOD females. These results open major therapeutic perspectives. They strongly suggest that self-tolerance can be restored in adult mice once autoimmunity is fully established and confirm that this effect can be obtained by transient targeting of the CD3/T-cell receptor without massive T-celi debulking.

Regulatory T cells (T reg cells) play a major role in controlling the pathogenic autoimmune process in type 1 diabetes (T1D). Interleukin 2 (IL-2), a cytokine which promotes T reg cell survival and function, may thus have therapeutic efficacy in T1D. We show that 5 d of low-dose IL-2 administration starting at the time of T1D onset can reverse established disease in NOD (nonobese diabetic) mice, with long-lasting effects. Low-dose IL-2 increases the number of T reg cells in the pancreas and induces expression of T reg cell–associated proteins including Foxp3, CD25, CTLA-4, ICOS (inducible T cell costimulator), and GITR (glucocorticoid-induced TNF receptor) in these cells. Treatment also suppresses interferon γ production by pancreas-infiltrating T cells. Transcriptome analyses show that low-dose IL-2 exerts much greater influence on gene expression of T reg cells than effector T cells (T eff cells), suggesting that nonspecific activation of pathogenic T eff cells is less likely. We provide the first preclinical data showing that low-dose IL-2 can reverse established T1D, suggesting that this treatment merits evaluation in patients with T1D.

Anti-CD20 monoclonal antibodies are widely used for the treatment of hematological malignancies or autoimmune disease but may be responsible for a secondary humoral deficiency. In the context of COVID-19 infection, this may prevent the elicitation of a specific SARS-CoV-2-antibody response. We report a series of 17 consecutive patients with profound B-cell lymphopenia and prolonged COVID-19 symptoms, negative IgG-IgM SARS-CoV-2 serology and a positive RNAemia measured by digital PCR who were treated with four units of COVID-19 convalescent plasma. Within 48 hours following transfusion, all patients except one experienced an amelioration of their clinical symptoms. The inflammatory syndrome abated within a week. Only one patient who needed mechanical ventilation for severe COVID-19 disease died of bacterial pneumonia. SARS-CoV-2 RNAemia decreased to below the sensitivity threshold in 9 out of 9 evaluated patients. Analysis of virus-specific T-cell responses using T-cell enzyme linked immunoSpot (ELISPOT) assay was analyzed before convalescent plasma transfusion in 3 patients. All showed a conserved SARS-CoV-2 T-cell response and poor cross-response to other coronaviruses. No adverse event was reported. In COVID-19 patients unable to mount a specific humoral response to SARS-CoV-2, convalescent plasma with anti-SARS-CoV-2 antibodies appears to be a very promising approach in the context of protracted COVID-19 symptoms.

Targeted immunotherapies hold great promise for the treatment and cure of autoimmune diseases. The efficacy of CD3-specific monoclonal antibody therapy in mice and humans stems from its ability to re-establish immune homeostasis in treated individuals. This occurs through modulation of the T-cell receptor (TCR)-CD3 complex (also termed antigenic modulation) and/or induction of apoptosis of activated autoreactive T cells, which leaves behind 'space' for homeostatic reconstitution that favours selective induction, survival and expansion of adaptive regulatory T cells, which establishes long-term tolerance. This Review summarizes the pre-clinical and clinical studies of CD3-specific monoclonal antibody therapy and highlights future opportunities to enhance the efficacy of this potent immunotherapeutic.

Aims/hypothesis The aim of the study was to examine the 48 month outcome of treating recent-onset type 1 diabetic patients for 6 days with humanised CD3-antibody, ChAglyCD3. Methods Eighty patients, aged 12-39 years, were recruited for a phase 2 multicentre trial and randomised to placebo (n=40) or ChAglyCD3 (n=40) treatment by a third party member; participants and care-givers were blinded. The change in insulin dose (U kg −1 day −1 ) over 48 months was chosen as primary endpoint and compared in 31 placeboand 33 ChAglyCD3-treated patients. Adverse events were followed in 35 and 38 patients, respectively. Results Treatment with ChAglyCD3 delayed the rise in insulin requirements of patients with recent-onset diabetes and reduced its amplitude over 48 months (+0.09 vs +0.32 U kg −1 day −1 in the placebo group). Using multivariate analysis this effect was correlated with higher baseline residual beta cell function and a younger age. It was associated with better outcome variables in subgroups selected according to these variables. In the ChAglyCD3 subgroup with higher initial beta cell function, 0/11 patients became C-peptide-negative over 48 months vs 4/9 in the corresponding placebo subgroup. In the subgroup aged <27 years old, antibody treatment preserved initial beta cell function for 36 months (vs >80% decline within 24 months in the placebo subgroup <27 years old), resulted in lower HbA 1c concentrations and tended to reduce glycaemic variability (p=0.08). No longterm adverse events were observed. Conclusions/interpretation A 6 day ChAglyCD3 treatment can suppress the rise in insulin requirements of recent-onset type 1 diabetic patients over 48 months, depending on their age and initial residual beta cell function. In younger patients this effect is associated with reduced deterioration of metabolic variables. These observations help to define inclusion criteria for prevention trials.