Single-center randomized trial of T-reg graft alone vs T-reg graft plus tacrolimus for the prevention of acute GVHD.

ABSTRACT: Allogeneic hematopoietic cell transplantation (HCT) is a curative therapy for hematological malignancies for which graft-versus-host disease (GVHD) remains a major complication. The use of donor T-regulatory cells (Tregs) to prevent GVHD appears promising, including in our previous evaluation of an engineered graft product (T-reg graft) consisting of the timed, sequential infusion of CD34+ hematopoietic stem cells and high-purity Tregs followed by conventional T cells. However, whether immunosuppressive prophylaxis can be removed from this protocol remains unclear. We report the results of the first stage of an open-label single-center phase 2 study (NCT01660607) investigating T-reg graft in myeloablative HCT of HLA-matched and 9/10-matched recipients. Twenty-four patients were randomized to receive T-reg graft alone (n = 12) or T-reg graft plus single-agent GVHD prophylaxis (n = 12) to determine whether T-reg graft alone was noninferior in preventing acute GVHD. All patients developed full-donor myeloid chimerism. Patients with T-reg graft alone vs with prophylaxis had incidences of grade 3 to 4 acute GVHD of 58% vs 8% (P = .005) and grade 3 to 4 of 17% vs 0% (P = .149), respectively. The incidence of moderate-to-severe chronic GVHD was 28% in the T-reg graft alone arm vs 0% with prophylaxis (P = .056). Among patients with T-reg graft and prophylaxis, CD4+ T-cell-to-Treg ratios were reduced after transplantation, gene expression profiles showed reduced CD4+ proliferation, and the achievement of full-donor T-cell chimerism was delayed. This study indicates that T-reg graft with single-agent tacrolimus is preferred over T-reg graft alone for the prevention of acute GVHD. This trial was registered at www.clinicaltrials.gov as #NCT01660607.

Selective decrease of donor-reactive Tregs after liver transplantation limits Treg therapy for promoting allograft tolerance in humans.

Promoting immune tolerance to transplanted organs can minimize the amount of immunosuppressive drugs that patients need to take, reducing lifetime risks of mortality and morbidity. Regulatory T cells (Tregs) are essential for immune tolerance, and preclinical studies have shown their therapeutic efficacy in inducing transplantation tolerance. Here, we report the results of a phase 1/2 trial (ARTEMIS, NCT02474199) of autologous donor alloantigen-reactive Treg (darTreg) therapy in individuals 2 to 6 years after receiving a living donor liver transplant. The primary efficacy endpoint was calcineurin inhibitor dose reduction by 75% with stable liver function tests for at least 12 weeks. Among 10 individuals who initiated immunosuppression withdrawal, 1 experienced rejection before planned darTreg infusion, 5 received darTregs, and 4 were not infused because of failure to manufacture the minimal infusible dose of 100 × 106 cells. darTreg infusion was not associated with adverse events. Two darTreg-infused participants reached the primary endpoint, but an insufficient number of recipients were treated for assessing the efficacy of darTregs. Mechanistic studies revealed generalized Treg activation, senescence, and selective reduction of donor reactivity after liver transplantation. Overall, the ARTEMIS trial features a design concept for evaluating the efficacy of Treg therapy in transplantation. The mechanistic insight gained from the study may help guide the design of future trials.

Undergraduate Supervised Clinical Practicum Activities: An Enlightening Exploration.

BACKGROUND: Clinical nursing practice is the hallmark of nursing education providing for the application of nursing knowledge to the care of patients in a contextual clinical environment. There is no universal method for educating students in the clinical arena. The literature has been limited to the evaluation of clinical education models and student perceptions of learning; however, there is a gap in the literature regarding the daily clinical activities of faculty and students. METHOD: This exploratory descriptive study examined the explicit undertakings of a clinical day among faculty and students in the southeastern United States. RESULTS: Responses from 61 survey participants described detailed activities of a clinical day including preclinical preparation, prebriefing, student and faculty clinical activities, and postconference structure. CONCLUSION: This foundational knowledge provides insight for improving clinical education with the goal of educators connecting clinical activities to the development of student competencies. [J Nurs Educ. 2022;61(10):591-593.].

Polyclonal Regulatory T Cell Manufacturing Under cGMP: A Decade of Experience.

We report on manufacturing outcomes for 41 autologous polyclonal regulatory T cell (PolyTreg) products for 7 different Phase 1 clinical trials over a 10-year period (2011-2020). Data on patient characteristics, manufacturing parameters, and manufacturing outcomes were collected from manufacturing batch records and entered into a secure database. Overall, 88% (36/41) of PolyTreg products met release criteria and 83% (34/41) of products were successfully infused into patients. Of the 7 not infused, 5 failed release criteria, and 2 were not infused because the patient became ineligible due to a change in clinical status. The median fold expansion over the 14-day manufacturing process was 434.8 -fold (range 29.8-2,232), resulting in a median post-expansion cell count of 1,841 x 106 (range 56.9-16,179 x 106). The main correlate of post-expansion cell number was starting cell number, which positively correlates with absolute circulating Treg cell count. Other parameters, including date of PolyTreg production, patient sex, and patient age did not significantly correlate with fold expansion of Treg during product manufacturing. In conclusion, PolyTreg manufacturing outcomes are consistent across trials and dates of production.

The effect of low-dose IL-2 and Treg adoptive cell therapy in patients with type 1 diabetes.

BACKGROUNDA previous phase I study showed that the infusion of autologous Tregs expanded ex vivo into patients with recent-onset type 1 diabetes (T1D) had an excellent safety profile. However, the majority of the infused Tregs were undetectable in the peripheral blood 3 months postinfusion (Treg-T1D trial). Therefore, we conducted a phase I study (TILT trial) combining polyclonal Tregs and low-dose IL-2, shown to enhance Treg survival and expansion, and assessed the impact over time on Treg populations and other immune cells.METHODSPatients with T1D were treated with a single infusion of autologous polyclonal Tregs followed by one or two 5-day courses of recombinant human low-dose IL-2 (ld-IL-2). Flow cytometry, cytometry by time of flight, and 10x Genomics single-cell RNA-Seq were used to follow the distinct immune cell populations' phenotypes over time.RESULTSMultiparametric analysis revealed that the combination therapy led to an increase in the number of infused and endogenous Tregs but also resulted in a substantial increase from baseline in a subset of activated NK, mucosal associated invariant T, and clonal CD8+ T cell populations.CONCLUSIONThese data support the hypothesis that ld-IL-2 expands exogenously administered Tregs but also can expand cytotoxic cells. These results have important implications for the use of a combination of ld-IL-2 and Tregs for the treatment of autoimmune diseases with preexisting active immunity.TRIAL REGISTRATIONClinicalTrials.gov NCT01210664 (Treg-T1D trial), NCT02772679 (TILT trial).FUNDINGSean N. Parker Autoimmune Research Laboratory Fund, National Center for Research Resources.

Adoptive Treg Cell Therapy in a Patient With Systemic Lupus Erythematosus.

OBJECTIVE: Adoptive Treg cell therapy has great potential to treat autoimmune disease. Currently, very little is known about how these cells impact inflamed tissues. This study was undertaken to elucidate how autologous Treg cell therapy influences tissue inflammation in human autoimmune disease. METHODS: We describe a systemic lupus erythematosus (SLE) patient with active skin disease who received adoptive Treg therapy. We comprehensively quantified Treg cells and immune activation in peripheral blood and skin, with data obtained at multiple time points posttreatment. RESULTS: Deuterium tracking of infused Treg cells revealed the transient presence of cells in peripheral blood, accompanied by increased percentages of highly activated Treg cells in diseased skin. Flow cytometric analysis and whole transcriptome RNA sequencing revealed that Treg cell accumulation in skin was associated with a marked attenuation of the interferon-γ pathway and a reciprocal augmentation of the interleukin-17 (IL-17) pathway. This phenomenon was more pronounced in skin relative to peripheral blood. To validate these findings, we investigated Treg cell adoptive transfer of skin inflammation in a murine model and found that it also resulted in a pronounced skewing away from Th1 immunity and toward IL-17 production. CONCLUSION: We report the first case of a patient with SLE treated with autologous adoptive Treg cell therapy. Taken together, our results suggest that this treatment leads to increased activated Treg cells in inflamed skin, with a dynamic shift from Th1 to Th17 responses.

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

Reforming the undergraduate nursing clinical curriculum through clinical immersion: A literature review.

Clinical immersion is a method used by various academic programs to narrow the theory-to-practice gap and assist students to transition from school to a new work environment. In the clinical immersion model, students embark upon a concentrated and intensive clinical experience, typically at the end of a semester or program. This literature review explored the various methods by which programs carry out the immersion clinical experience model and if the experience improved students' readiness for entry level positions. Findings from students, faculty, and preceptors showed that immersion experiences are successful in increasing student confidence and nursing skills; however, additional objective evidence is needed to show that the use of immersion experiences can improve graduate readiness for practice. Research is also needed to explore if any differences in student performance outcomes exist between clinical immersion at the end of each semester versus one in a capstone course.

Expansion of Human Tregs from Cryopreserved Umbilical Cord Blood for GMP-Compliant Autologous Adoptive Cell Transfer Therapy.

Umbilical cord blood is a traditional and convenient source of cells for hematopoietic stem cell transplantation. Thymic regulatory T cells (Tregs) are also present in cord blood, and there is growing interest in the use of autologous Tregs to provide a low-risk, fully human leukocyte antigen (HLA)-matched cell product for treating autoimmune diseases, such as type 1 diabetes. Here, we describe a good manufacturing practice (GMP)-compatible Treg expansion protocol using fluorescence-activated cell sorting, resulting in a mean 2,092-fold expansion of Tregs over a 16-day culture for a median yield of 1.26 × 109 Tregs from single-donor cryopreserved units. The resulting Tregs passed prior clinical trial release criteria for Treg purity and sterility, including additional rigorous assessments of FOXP3 and Helios expression and epigenetic analysis of the FOXP3 Treg-specific demethylated region (TSDR). Compared with expanded adult peripheral blood Tregs, expanded cord blood Tregs remained more naive, as assessed by continued expression of CD45RA, produced reduced IFN-γ following activation, and effectively inhibited responder T cell proliferation. Immunosequencing of the T cell receptor revealed a remarkably diverse receptor repertoire within cord blood Tregs that was maintained following in vitro expansion. These data support the feasibility of generating GMP-compliant Tregs from cord blood for adoptive cell transfer therapies and highlight potential advantages in terms of safety, phenotypic stability, autoantigen specificity, and tissue distribution.

Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization.

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.

Type 1 diabetes immunotherapy using polyclonal regulatory T cells.

Type 1 diabetes (T1D) is an autoimmune disease that occurs in genetically susceptible individuals. Regulatory T cells (Tregs) have been shown to be defective in the autoimmune disease setting. Thus, efforts to repair or replace Tregs in T1D may reverse autoimmunity and protect the remaining insulin-producing ß cells. On the basis of this premise, a robust technique has been developed to isolate and expand Tregs from patients with T1D. The expanded Tregs retained their T cell receptor diversity and demonstrated enhanced functional activity. We report on a phase 1 trial to assess safety of Treg adoptive immunotherapy in T1D. Fourteen adult subjects with T1D, in four dosing cohorts, received ex vivo-expanded autologous CD4(+)CD127(lo/-)CD25(+) polyclonal Tregs (0.05 × 10(8) to 26 × 10(8) cells). A subset of the adoptively transferred Tregs was long-lived, with up to 25% of the peak level remaining in the circulation at 1 year after transfer. Immune studies showed transient increases in Tregs in recipients and retained a broad Treg FOXP3(+)CD4(+)CD25(hi)CD127(lo) phenotype long-term. There were no infusion reactions or cell therapy-related high-grade adverse events. C-peptide levels persisted out to 2+ years after transfer in several individuals. These results support the development of a phase 2 trial to test efficacy of the Treg therapy.

Serum IgE clearance is facilitated by human FcεRI internalization.

The high-affinity IgE receptor FcεRI is constitutively expressed in mast cells and basophils and is required for transmitting stimulatory signals upon engagement of IgE-bound allergens. FcεRI is also constitutively expressed in dendritic cells (DCs) and monocytes in humans; however, the specific functions of the FcεRI expressed by these cells are not completely understood. Here, we found that FcεRI expressed by human blood DC antigen 1-positive (BDCA1+) DCs and monocytes, but not basophils, traffics to endolysosomal compartments under steady-state conditions. Furthermore, IgE bound to FcεRI on BDCA1+ DCs was rapidly endocytosed, transported to the lysosomes, and degraded in vitro. IgE injected into mice expressing human FcεRIα (FCER1A-Tg mice) was endocytosed by conventional DCs and monocytes, and endocytosis was associated with rapid clearance of circulating IgE from these mice. Importantly, this rapid IgE clearance was dependent on monocytes or DCs but not basophils. These findings strongly suggest that constitutive internalization of human FcεRI by DCs and monocytes distinctively contributes to serum IgE clearance.

The Net Generation of nursing: keeping empathetic communication alive.

Electronic communication has had a profound impact on generations, in the nursing profession as well as in society as a whole. Nursing educators struggle with facilitating verbal communication skills in didactic and clinical settings, particularly with the Net Generation. Online education is rapidly becoming the norm in degree-completion programs. Nursing educators must assure that empathetic communication with patients will not become a lost art.

Rapid assessment of in vitro expanded human regulatory T cell function.

Human regulatory T cells (Treg) are able to actively suppress autoreactive immune responses. Phenotypically, they are broadly characterized as CD4+, CD25+, CD127(lo/⁻) and FoxP3+. CD45RA can be used to further differentiate the population into naïve (CD45RA(+)) and induced (CD45RA⁻) Treg. The functional potential of Treg is routinely determined by assessing their ability to suppress T cell function in 3-5day proliferation assays. Since Treg are being explored for therapeutic use, a short-term functional assay could serve as a valuable tool for evaluating the potency of Treg. Therefore, an assay designed to measure Treg suppression of activation marker expression by responder T cells in 7 to 20h has been examined in this report. Using flow cytometry, expression of CD69 and CD154 on T cells, in response to stimulation with CD3/CD28 beads, was used as a measure of activation in the assay. Treg from healthy volunteers were sorted as CD4+CD25+CD127(lo/⁻)CD45RA+ cells with a BD FACSAria™ II. The highly purified Treg were then expanded in vitro and their function was assessed in short term activation marker suppression assays using autologous PBMC as responder cells. The data suggest that this short term suppression assay could be a reliable surrogate for assessing Treg functional potential.

Plasticity of human regulatory T cells in healthy subjects and patients with type 1 diabetes.

Regulatory T cells (Tregs) constitute an attractive therapeutic target given their essential role in controlling autoimmunity. However, recent animal studies provide evidence for functional heterogeneity and lineage plasticity within the Treg compartment. To understand better the plasticity of human Tregs in the context of type 1 diabetes, we characterized an IFN-γ-competent subset of human CD4(+)CD127(lo/-)CD25(+) Tregs. We measured the frequency of Tregs in the peripheral blood of patients with type 1 diabetes by epigenetic analysis of the Treg-specific demethylated region (TSDR) and the frequency of the IFN-γ(+) subset by flow cytometry. Purified IFN-γ(+) Tregs were assessed for suppressive function, degree of TSDR demethylation, and expression of Treg lineage markers FOXP3 and Helios. The frequency of Tregs in peripheral blood was comparable but the FOXP3(+)IFN-γ(+) fraction was significantly increased in patients with type 1 diabetes compared to healthy controls. Purified IFN-γ(+) Tregs expressed FOXP3 and possessed suppressive activity but lacked Helios expression and were predominately methylated at the TSDR, characteristics of an adaptive Treg. Naive Tregs were capable of upregulating expression of Th1-associated T-bet, CXCR3, and IFN-γ in response to IL-12. Notably, naive, thymic-derived natural Tregs also demonstrated the capacity for Th1 differentiation without concomitant loss of Helios expression or TSDR demethylation.

Human antigen-specific regulatory T cells generated by T cell receptor gene transfer.

BACKGROUND: Therapies directed at augmenting regulatory T cell (Treg) activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects, including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments, with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However, current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover, FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific, whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition. METHODOLOGY/PRINCIPAL FINDINGS: To overcome these limitations, we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR) gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs, and maintained the capacity to suppress conventional T cell responses directed against tyrosinase, as well as bystander T cell responses. Using this methodology in a model tumor system, murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff) activity as determined by tumor cell growth and luciferase reporter-based imaging. CONCLUSIONS/SIGNIFICANCE: These results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy.

The role of X-linked FOXP3 in the autoimmune susceptibility of Turner Syndrome patients.

Turner Syndrome patients have an absent second sex chromosome and a predisposition to autoimmune disease. We hypothesized that the autoimmune susceptibility in Turner Syndrome may be due to an alteration in the expression of the X-linked FOXP3 gene. FOXP3 is important in the development of regulatory T cells, and complete loss of FOXP3 expression has been shown to result in severe autoimmunity. To test this hypothesis, we characterized the regulatory T cells and performed immunophenotyping on the peripheral blood leukocytes of a cohort of Turner Syndrome patients. These patients retained regulatory T cell frequency and function despite an increased prevalence of autoimmunity. Immunophenotyping revealed a decrease in the ratio of CD4 to CD8 lymphocytes. These findings suggest that the autoimmune predisposition in Turner Syndrome is not due to alterations in regulatory T cells but may be associated with a change in the proportion of T cell subsets.

Expansion of human regulatory T-cells from patients with type 1 diabetes.

OBJECTIVE: Regulatory T-cells (Tregs) have catalyzed the field of immune regulation. However, translating Treg-based therapies from animal models of autoimmunity to human clinical trials requires robust methods for the isolation and expansion of these cells-a need forming the basis for these studies. RESEARCH DESIGN AND METHODS: Tregs from recent-onset type 1 diabetic patients and healthy control subjects were isolated by fluorescence-activated cell sorting and compared for their capacity to expand in vitro in response to anti-CD3-anti-CD28-coated microbeads and IL-2. Expanded cells were examined for suppressive function, lineage markers and FOXP3, and cytokine production. RESULTS: Both CD4+CD127(lo/-) and CD4+CD127(lo/-)CD25+ T-cells could be expanded and used as Tregs. However, expansion of CD4+CD127(lo/-) cells required the addition of rapamycin to maintain lineage purity. In contrast, expansion of CD4+CD127(lo/-)CD25+ T-cells, especially the CD45RA+ subset, resulted in high yield, functional Tregs that maintained higher FOXP3 expression in the absence of rapamycin. Tregs from type 1 diabetic patients and control subjects expanded similarly and were equally capable of suppressing T-cell proliferation. Regulatory cytokines were produced by Tregs after culture; however, a portion of FOXP3+ cells were capable of producing interferon (IFN)-gamma after reactivation. IFN-gamma production was observed from both CD45RO+ and CD45RA+ Treg populations. CONCLUSIONS: The results support the feasibility of isolating Tregs for in vitro expansion. Based on expansion capacity, FOXP3 stability, and functional properties, the CD4+CD127(lo/-)CD25+ T-cells represent a viable cell population for cellular therapy in this autoimmune disease.

Human regulatory T cells: role in autoimmune disease and therapeutic opportunities.

SUMMARY: The importance of regulatory T lymphocytes (Tregs) in the control of autoimmunity is now well established in a variety of experimental animal models. In addition, there are numerous studies suggesting that Treg deficits may be an underlying cause of human autoimmune diseases. The emergence of Tregs as an essential component of immune homeostasis provides a potential therapeutic opportunity for active immune regulation and long-term tolerance induction. In this article, we summarize the core basic science and animal model studies of Tregs, review the status of multiple biologic and small molecule chemical compounds to promote Treg development in vivo, and discuss recent advances for the identification and expansion of polyclonal and antigen-specific Tregs for adoptive immunotherapy. In summary, the review provides an in-depth analysis and highlights the challenges and opportunities for immune intervention with Treg-based therapeutics.

A unique T cell subset described as CD4loCD40+ T cells (TCD40) in human type 1 diabetes.

Human T1D pancreatic lymph nodes contain diabetes-autoantigen responsive T cells but identification of such T cells in the periphery has proven difficult. Here we describe a unique T cell subset defined by CD4(lo) and CD40 expression (T(CD40)) that is significantly expanded in peripheral blood of T1D but not control or T2D subjects. The HLA-DR3 and DR4 alleles are considered high risk factors for T1D and T(CD40) expansion occurs in T1D subjects carrying HLA DR3 or DR4 haplotypes but, T1D subjects who do not carry either DR3 or DR4 haplotypes still have an expanded percentage of T(CD40) cells. Non-autoimmune subjects, even DR3(+) and DR4(+), do not have elevated percentages of T(CD40) cells. The majority of T(CD40) cells in T1D carry a memory phenotype and a portion of those proliferates when exposed to diabetes-associated self-antigens. A greater number of memory T(CD40) cells express CXCR3 when compared to CD40(-) memory cells and that number is significantly expanded in T1D compared to control subjects. If only total CD4(+) T cells are compared no difference in CXCR3 is seen. Furthermore, T(CD40) cells produce a Th1, pro-inflammatory cytokine profile. In healthy controls, T(CD40) cells have equally Th1 and Th2 profiles.