Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency.

We previously reported two siblings with inherited PD-1 deficiency who died from autoimmune pneumonitis at 3 and 11 years of age after developing other autoimmune manifestations, including type 1 diabetes (T1D). We report here two siblings, aged 10 and 11 years, with neonatal-onset T1D (diagnosed at the ages of 1 day and 7 wk), who are homozygous for a splice-site variant of CD274 (encoding PD-L1). This variant results in the exclusive expression of an alternative, loss-of-function PD-L1 protein isoform in overexpression experiments and in the patients' primary leukocytes. Surprisingly, cytometric immunophenotyping and single-cell RNA sequencing analysis on blood leukocytes showed largely normal development and transcriptional profiles across lymphoid and myeloid subsets in the PD-L1-deficient siblings, contrasting with the extensive dysregulation of both lymphoid and myeloid leukocyte compartments in PD-1 deficiency. Our findings suggest that PD-1 and PD-L1 are essential for preventing early-onset T1D but that, unlike PD-1 deficiency, PD-L1 deficiency does not lead to fatal autoimmunity with extensive leukocytic dysregulation.

Intratumoral pan-ErbB targeted CAR-T for head and neck squamous cell carcinoma: interim analysis of the T4 immunotherapy study.

BACKGROUND: Locally advanced/recurrent head and neck squamous cell carcinoma (HNSCC) is associated with significant morbidity and mortality. To target upregulated ErbB dimer expression in this cancer, we developed an autologous CD28-based chimeric antigen receptor T-cell (CAR-T) approach named T4 immunotherapy. Patient-derived T-cells are engineered by retroviral transduction to coexpress a panErbB-specific CAR called T1E28ζ and an IL-4-responsive chimeric cytokine receptor, 4αß, which allows IL-4-mediated enrichment of transduced cells during manufacture. These cells elicit preclinical antitumor activity against HNSCC and other carcinomas. In this trial, we used intratumoral delivery to mitigate significant clinical risk of on-target off-tumor toxicity owing to low-level ErbB expression in healthy tissues. METHODS: We undertook a phase 1 dose-escalation 3+3 trial of intratumoral T4 immunotherapy in HNSCC (NCT01818323). CAR T-cell batches were manufactured from 40 to 130 mL of whole blood using a 2-week semiclosed process. A single CAR T-cell treatment, formulated as a fresh product in 1-4 mL of medium, was injected into one or more target lesions. Dose of CAR T-cells was escalated in 5 cohorts from 1×107-1×109 T4+ T-cells, administered without prior lymphodepletion. RESULTS: Despite baseline lymphopenia in most enrolled subjects, the target cell dose was successfully manufactured in all cases, yielding up to 7.5 billion T-cells (67.5±11.8% transduced), without any batch failures. Treatment-related adverse events were all grade 2 or less, with no dose-limiting toxicities (Common Terminology Criteria for Adverse Events V.4.0). Frequent treatment-related adverse events were tumor swelling, pain, pyrexias, chills, and fatigue. There was no evidence of leakage of T4+ T-cells into the circulation following intratumoral delivery, and injection of radiolabeled cells demonstrated intratumoral persistence. Despite rapid progression at trial entry, stabilization of disease (Response Evaluation Criteria in Solid Tumors V.1.1) was observed in 9 of 15 subjects (60%) at 6 weeks post-CAR T-cell administration. Subsequent treatment with pembrolizumab and T-VEC oncolytic virus achieved a rapid complete clinical response in one subject, which was durable for over 3 years. Median overall survival was greater than for historical controls. Disease stabilization was associated with the administration of an immunophenotypically fitter, less exhausted, T4 CAR T-cell product. CONCLUSIONS: These data demonstrate the safe intratumoral administration of T4 immunotherapy in advanced HNSCC.

Monitoring islet specific immune responses in type 1 diabetes clinical immunotherapy trials.

The number of immunotherapeutic clinical trials in type 1 diabetes currently being conducted is expanding, and thus there is a need for robust immune-monitoring assays which are capable of detecting and characterizing islet specific immune responses in peripheral blood. Islet- specific T cells can serve as biomarkers and as such can guide drug selection, dosing regimens and immunological efficacy. Furthermore, these biomarkers can be utilized in patient stratification which can then benchmark suitability for participation in future clinical trials. This review focusses on the commonly used immune-monitoring techniques including multimer and antigen induced marker assays and the potential to combine these with single cell transcriptional profiling which may provide a greater understanding of the mechanisms underlying immuno-intervention. Although challenges remain around some key areas such as the need for harmonizing assays, technological advances mean that multiparametric information derived from a single sample can be used in coordinated efforts to harmonize biomarker discovery and validation. Moreover, the technologies discussed here have the potential to provide a unique insight on the effect of therapies on key players in the pathogenesis of T1D that cannot be obtained using antigen agnostic approaches.

Low dose interleukin-2 selectively expands circulating regulatory T cells but fails to promote liver allograft tolerance in humans.

BACKGROUND & AIMS: CD4+CD25+Foxp3+ regulatory T cells (Tregs) are essential to maintain immunological tolerance and have been shown to promote liver allograft tolerance in both rodents and humans. Low-dose IL-2 (LDIL-2) can expand human endogenous circulating Tregs in vivo, but its role in suppressing antigen-specific responses and promoting Treg trafficking to the sites of inflammation is unknown. Likewise, whether LDIL-2 facilitates the induction of allograft tolerance has not been investigated in humans. METHODS: We conducted a clinical trial in stable liver transplant recipients 2-6 years post-transplant to determine the capacity of LDIL-2 to suppress allospecific immune responses and allow for the complete discontinuation of maintenance immunosuppression (ClinicalTrials.gov NCT02949492). One month after LDIL-2 was initiated, those exhibiting at least a 2-fold increase in circulating Tregs gradually discontinued immunosuppression over a 4-month period while continuing LDIL-2 for a total treatment duration of 6 months. RESULTS: All participants achieved a marked and sustained increase in circulating Tregs. However, this was not associated with the preferential expansion of donor-reactive Tregs and did not promote the accumulation of intrahepatic Tregs. Furthermore, LDIL-2 induced a marked IFNγ-orchestrated transcriptional response in the liver even before immunosuppression weaning was initiated. The trial was terminated after the first 6 participants failed to reach the primary endpoint owing to rejection requiring reinstitution of immunosuppression. CONCLUSIONS: The expansion of circulating Tregs in response to LDIL-2 is not sufficient to control alloimmunity and to promote liver allograft tolerance, due, at least in part, to off-target effects that increase liver immunogenicity. Our trial provides unique insight into the mechanisms of action of immunomodulatory therapies such as LDIL-2 and their limitations in promoting alloantigen-specific effects and immunological tolerance. CLINICAL TRIALS REGISTRATION: The study is registered at ClinicalTrials.gov (NCT02949492). IMPACT AND IMPLICATIONS: The administration of low-dose IL-2 is an effective way of increasing the number of circulating regulatory T cells (Tregs), an immunosuppressive lymphocyte subset that is key for the establishment of immunological tolerance, but its use to promote allograft tolerance in the setting of clinical liver transplantation had not been explored before. In liver transplant recipients on tacrolimus monotherapy, low-dose IL-2 effectively expanded circulating Tregs but did not increase the number of Tregs with donor specificity, nor did it promote their trafficking to the transplanted liver. Low-dose IL-2 did not facilitate the discontinuation of tacrolimus and elicited, as an off-target effect, an IFNγ-orchestrated inflammatory response in the liver that resembled T cell-mediated rejection. These results, supporting an unexpected role for IL-2 in regulating the immunogenicity of the liver, highlight the need to carefully evaluate systemic immunoregulatory strategies with investigations that are not restricted to the blood compartment and involve target tissues such as the liver.

Immune and Metabolic Effects of Antigen-Specific Immunotherapy Using Multiple ß-Cell Peptides in Type 1 Diabetes.

Type 1 diabetes is characterized by a loss of tolerance to pancreatic ß-cell autoantigens and defects in regulatory T-cell (Treg) function. In preclinical models, immunotherapy with MHC-selective, autoantigenic peptides restores immune tolerance, prevents diabetes, and shows greater potency when multiple peptides are used. To translate this strategy into the clinical setting, we administered a mixture of six HLA-DRB1*0401-selective, ß-cell peptides intradermally to patients with recent-onset type 1 diabetes possessing this genotype in a randomized placebo-controlled study at monthly doses of 10, 100, and 500 µg for 24 weeks. Stimulated C-peptide (measuring insulin functional reserve) had declined in all placebo subjects at 24 weeks but was maintained at ≥100% baseline levels in one-half of the treated group. Treatment was accompanied by significant changes in islet-specific immune responses and a dose-dependent increase in Treg expression of the canonical transcription factor FOXP3 and changes in Treg gene expression. In this first-in-human study, multiple-peptide immunotherapy shows promise as a strategy to correct immune regulatory defects fundamental to the pathobiology of autoimmune diabetes.

A phase 1b open-label dose-finding study of ustekinumab in young adults with type 1 diabetes.

OBJECTIVES: We assessed the safety of ustekinumab (a monoclonal antibody used in psoriasis to target the IL-12 and IL-23 pathways) in a small cohort of recent-onset (<100 days of diagnosis) adults with type 1 diabetes (T1D) by conducting a pilot open-label dose-finding and mechanistic study (NCT02117765) at the University of British Columbia. METHODS: We sequentially enrolled 20 participants into four subcutaneous dosing cohorts: (i) 45 mg loading weeks 0/4/16, (ii) 45 mg maintenance weeks 0/4/16/28/40, (iii) 90 mg loading weeks 0/4/16, and (iv) 90 mg maintenance weeks 0/4/16/28/40. The primary endpoint was safety as assessed by an independent data and safety monitoring board (DSMB) but we also measured mixed meal tolerance test C-peptide, insulin use/kg, and HbA1c. Immunophenotyping was performed to assess immune cell subsets and islet antigen-specific T cell responses. RESULTS: Although several adverse events were reported, only two (bacterial vaginosis and hallucinations) were thought to be possibly related to drug administration by the study investigators. At 1 year, the 90 mg maintenance dosing cohort had the smallest mean decline in C-peptide area under the curve (AUC) (0.1 pmol/ml). Immunophenotyping showed that ustekinumab reduced the percentage of circulating Th17, Th1, and Th17.1 cells and proinsulin-specific T cells that secreted IFN-γ and IL-17A. CONCLUSION: Ustekinumab was deemed safe to progress to efficacy studies by the DSMB at doses used to treat psoriasis in adults with T1D. A 90 mg maintenance dosing schedule reduced proinsulin-specific IFN-γ and IL-17A-producing T cells. Further studies are warranted to determine if ustekinumab can prevent C-peptide AUC decline and induce a clinical response.

Mapping T Cell Responses to Native and Neo-Islet Antigen Epitopes in at Risk and Type 1 Diabetes Subjects.

Aims: Recent studies highlight the potentially important role of neoepitopes in breaking immune tolerance in type 1 diabetes. T cell reactivity to these neoepitopes has been reported, but how this response compares quantitatively and phenotypically with previous reports on native epitopes is not known. Thus, an understanding of the relationship between native and neoepitopes and their role as tolerance breakers or disease drivers in type 1 diabetes is required. We set out to compare T cell reactivity and phenotype against a panel of neo- and native islet autoantigenic epitopes to examine how this relates to stages of type 1 diabetes development. Methods: Fifty-four subjects comprising patients with T1D, and autoantibody-positive unaffected family members were tested against a panel of neo- and native epitopes by ELISPOT (IFN-γ, IL-10, and IL-17). A further subset of two patients was analyzed by Single Cell Immune Profiling (RNAseq and TCR α/ß) after stimulation with pools of native and neoepitope peptides. Results: T cell responses to native and neoepitopes were present in patients with type 1 diabetes and at-risk subjects, and overall, there were no significant differences in the frequency, magnitude, or phenotype between the two sets of peptide stimuli. Single cell RNAseq on responder T cells revealed a similar profile in T1D patients stimulated with either neo- or native epitopes. A pro-inflammatory gene expression profile (TNF-α, IFN-γ) was dominant in both native and neoepitope stimulated T cells. TCRs with identical clonotypes were found in T cell responding to both native and neoepitopes. Conclusion/Interpretation: These data suggest that in peripheral blood, T cell responses to both native and neoepitopes are similar in terms of frequency and phenotype in patients with type 1 diabetes and high-risk unaffected family members. Furthermore, using a combination of transcriptomic and clonotypic analyses, albeit using a limited panel of peptides, we show that neoepitopes are comparable to native epitopes currently in use for immune-monitoring studies.

Phenotypic Analysis of Human Lymph Nodes in Subjects With New-Onset Type 1 Diabetes and Healthy Individuals by Flow Cytometry.

Background: Ultrasound guided sampling of human lymph node (LN) combined with advanced flow cytometry allows phenotypic analysis of multiple immune cell subsets. These may provide insights into immune processes and responses to immunotherapies not apparent from analysis of the blood. Methods: Ultrasound guided inguinal LN samples were obtained by both fine needle aspiration (FNA) and core needle biopsy in 10 adults within 8 weeks of diagnosis of type 1 diabetes (T1D) and 12 age-matched healthy controls at two study centers. Peripheral blood mononuclear cells (PBMC) were obtained on the same occasion. Samples were transported same day to the central laboratory and analyzed by multicolour flow cytometry. Results: LN sampling was well-tolerated and yielded sufficient cells for analysis in 95% of cases. We confirmed the segregation of CD69+ cells into LN and the predominance of CD8+ Temra cells in blood previously reported. In addition, we demonstrated clear enrichment of CD8+ naïve, FOXP3+ Treg, class-switched B cells, CD56bright NK cells and plasmacytoid dendritic cells (DC) in LNs as well as CD4+ T cells of the Th2 phenotype and those expressing Helios and Ki67. Conventional NK cells were virtually absent from LNs as were Th22 and Th1Th17 cells. Paired correlation analysis of blood and LN in the same individuals indicated that for many cell subsets, especially those associated with activation: such as CD25+ and proliferating (Ki67+) T cells, activated follicular helper T cells and class-switched B cells, levels in the LN compartment could not be predicted by analysis of blood. We also observed an increase in Th1-like Treg and less proliferating (Ki67+) CD4+ T cells in LN from T1D compared to control LNs, changes which were not reflected in the blood. Conclusions: LN sampling in humans is well-tolerated. We provide the first detailed "roadmap" comparing immune subsets in LN vs. blood emphasizing a role for differentiated effector T cells in the blood and T cell regulation, B cell activation and memory in the LN. For many subsets, frequencies in blood, did not correlate with LN, suggesting that LN sampling would be valuable for monitoring immuno-therapies where these subsets may be impacted.