Precision Diagnostics in Inflammatory Bowel Disease, Cellular Therapy and Transplantation (The PREDICT Trial)

Hypotheses:

Hypothesis #1: The Investigators hypothesize that they can define the molecular mechanisms responsible for Inflammatory Bowel Disease (IBD) and gastrointestinal (GI) acute GVHD and differentiate it from other inflammatory disorders by using advanced immunologic analysis including flow cytometry, TCR deep sequencing and transcriptomics.

Hypothesis #2: The Investigators further hypothesize that longitudinal systems-based immunologic analysis will enable the patient-specific determination of the molecular evolution of IBD as well as acute and chronic GVHD as well post-transplant defects in protective immunity, and determine which pathways, when perturbed, can cause clinical disease. The discovery of these pathways will lead to improved diagnostic, prognostic and treatment approaches, and to personalized therapeutic decision-making for patients undergoing hematopoietic stem cell transplantation (HCT).

Hypothesis #3: We hypothesize that we can define the molecular mechanisms, phenotypic and functional immunologic characteristics involved in distinct determinants of adoptive cellular therapies, including the efficacy, longevity and toxicity associated with cellular therapy. Longitudinal characterization of cellular therapeutics and the endogenous immune response they elicit using advanced immunologic analysis including flow cytometry, mass spectrometry, TCR deep sequencing and single-cell transcriptomics will allow identification and distinction of pathways critical for efficacy and toxicity and enable subsequent therapeutic modulation.

Hypothesis #4: We hypothesize that differences in the gut microbiome of patients with IBD and recipients of HCT play a major role in disease severity and overall clinical outcomes in both diseases (e.g. bacteremia, unexplained fevers, mortality). The longitudinal characterization of the gut microbial communities by next generation sequencing will allow for detection of sequential microbial changes that coincide with observed clinical changes. the discovery of significant changes in the microbiome that are repeatedly observed with a particular clinical outcome will lead to better mechanistic understanding of its pathophysiology and inform future diagnostic and preventive approaches.

Hypothesis #5: We hypothesize that immune dysregulation associated with a wide range of disorders will alter the immune response to vaccines, compounding susceptibility to infectious diseases in these populations. Specifically, HCT and solid organ transplant recipients, as well as patients with active or recent history of malignancy, or autoimmune diseases may have reduced T and B cell responses to SARS-CoV-2 vaccination due to the effect of disease pathophysiology or treatment regimens on immune function. Comprehensive serologic analysis, high parameter flow cytometry, and T and B cell RNA sequencing will enable longitudinal analysis of neutralizing antibody titers and antigen-specific T and B cell expansion, phenotype, diversity, and survival following vaccination in these patient populations, as well as in related and unrelated healthy controls. These data will provide mechanistic insight into how immune impairment in these disorders contributes to poor responses to infections. as SARS-CoV-2 is a pathogen to which much of the population remains naïve, this represents a unique opportunity to study the immune response to a novel challenge in immunocompromised individuals. Moreover, these findings will inform public health guidelines on how to improve measures to protect vulnerable populations from preventable disease.

Aims:

Specific Aim #1: To identify the mechanisms specific for IBD and GI acute GVHD and delineate it from other inflammatory disorders.

Objective 1: Perform flow cytometry, TCR deep sequencing and whole transcriptome analysis on T cells purified from GI tissue samples taken from patients who undergo endoscopy for presumed GI GVHD, inflammatory bowel disease (IBD), and functional gastrointestinal disease (FGID).

Objective 2: Perform flow cytometry, TCR deep sequencing and transcriptome analysis on T cells from the peripheral blood at the time of endoscopy in patients diagnosed with GI GVHD, IBD, and FGID.

Specific Aim #2: Characterize the immunologic dysregulation responsible for IBD, acute GVHD, chronic GVHD and defects in protective immunity in patients undergoing HCT.

Objective 1: Perform longitudinal immune analysis on T cells and B cells purified from patients with IBD and those undergoing allogeneic HCT. For transplant patients, we will compare T and B cell immunity in patients who develop acute and chronic GVHD, relapse, and infectious complications post-transplant and compare to patients without these complications.

Objective 2: Perform microbiome analysis longitudinally in patients with IBD and those undergoing HCT to determine the impact of microbiome alterations in the development of post-transplant complications.

Specific Aim #3: Identify the molecular and cellular immunologic mechanisms involved in determining the clinical response to cellular therapies and distinguish pathways critical to a successful anti-tumor response from those involved in adverse effects.

Objective 1: Characterize the cellular product prior to administration and track its distribution, kinetics, persistence and function longitudinally in vivo in the patient's peripheral blood and when applicable bone marrow, CSF and other tissues, using qPCR-based transgene detection (if applicable), flow cytometry, mass cytometry, TCR deep sequencing and whole transcriptome analysis on T cells and other immune cells contained in the cellular product.

Objective 2: Longitudinally interrogate the interplay of the cellular therapy with the endogenous immune system and delineate the role of the endogenous immune response in the efficacy, persistence and toxicity of cellular therapy, using flow cytometry, mass cytometry, TCR deep sequencing, whole transcriptome analysis on endogenous immune cells and analysis of soluble factors and antibodies.

Specific Aim #4:Characterize the antigen-specific adaptive immune response to SARS-CoV-2 vaccination in patients with immune dysregulation due to cancer, transplantation, or autoimmune disease and identify mechanisms underlying impaired generation of durable immunity.

Objective 1: Perform comprehensive longitudinal analysis of serologic immunity pre- and post-vaccination, including to booster vaccines, in patients and healthy controls, including assessment of SARS-CoV-2 specific antibody levels and neutralizing antibody titers.

Objective 2: Use high parameter flow cytometry, single cell RNA sequencing (scRNAseq), and T and B cell repertoire analysis to characterize the longitudinal development of antigen-specific T and B cell memory to SARS-CoV-2 following vaccination and subsequent booster vaccines in patients and healthy controls.

Objective 3: Assess the ability of sera and cloned antibodies from patients to respond to bind and neutralize viral variants, as compared to that of healthy controls.

Background and Significance:

IBD: Inflammatory bowel disease (IBD) which includes Crohn's Disease (CD) and Ulcerative Colitis (UC), is a chronic complex gastrointestinal (GI) autoimmune condition that inflicts 1.4 million people in the united states1. The incidence and prevalence of both CD and UC are increasing over time and encompassing larger areas of the world1,2. In addition, pediatric IBD comprises 25% of all diagnosed IBD, relegating the child to a lifetime of gastrointestinal disease and exposure to immunosuppression especially during a period meant for growth and development. Despite ongoing research into the pathogenesis and genetic abnormalities, the mechanism behind IBD development and progression is not well understood. Standard therapies still rely on steroids, other non-specific immunosuppression (such as methotrexate and azathioprine), and anti-TNF biologics. Although newer therapies such as agents that block cytokines and leukocyte trafficking are emerging, no universally successful treatments have been identified. Thus, relapsing forms of IBD continue to lead to systemic compromise in nutritional absorptive capacity, anemia, and often, to the need for surgical interventions. Deciphering the mechanisms driving the unique subtypes of IBD (even within UC and CD) then optimizing treatment based on the underlying systemic dysregulation is a critical unmet need in the field. While the underlying immune mechanism of IBD remains undetermined, there is significant data to suggest that IBD may represent an inappropriate immune response towards self antigens and commensal microbiota in a genetically susceptible host3. Thus, murine colitis models suggest that mucosal inflammation results from pathologic T helper- (Th) cell responses, along with regulatory cell defects. These data have emerged from experiments in IL-2 deficient mice4, IL-10 deficient mice5, TGF-beta6, and TGF-betaGRII dominant negative transgenic mice7. The pathogenesis also includes an exaggeration in effector cell responses, which have emerged from experiments in Stat4 transgenic mice8 and TNFARE mutant mice9. More recently human cytokine analysis suggests that despite clinical similarities, each subtype of IBD show distinctive cytokine profiles10. Although initial studies have begun to target specific effector T cell pathways11, the application of target organ transcriptomics is in its infancy and individual targetable pathways are still elusive.

HCT: Allogeneic HCT is an effective treatment for patients with malignant and non-malignant hematologic diseases. However, this treatment is complicated with high rates of morbidity and mortality limiting its broader application. The leading causes of post-transplant morbidity and mortality include acute and chronic GVHD, relapse and infectious disease. The goal of the PREDICT trial is to apply a systems approach to understanding the mechanisms driving these complications, such that evidence-based treatment strategies can be devised.

Acute GVHD: Acute GVHD is mediated by donor-derived allo-reactive T cells becoming activated and resulting in cytotoxicity against host cells12,13 as well as cytokine-mediated tissue damage. Moderate to severe acute GVHD can occur in up to 60% of patients undergoing HCT and the more severe forms have been associated with mortality rates \>50%14-17. The most common sites of the immune-mediated tissue damage are the liver, skin, and gastrointestinal (GI) tract. GI GVHD occurs in 40-50% of HCT patients and is the major cause of morbidity and mortality from this disease17.

The diagnosis of GI GVHD is derived from clinical and histopathological findings. GVHD can occur in both the upper and lower GI tract leading to symptoms of diarrhea, abdominal pain, nausea, vomiting, and/or anorexia12. Histopathological diagnostic criteria for GI GVHD includes identification of crypt cell apoptosis, crypt destruction and/or mucosa denudation18. Unfortunately, the severity of GVHD on histology is poorly correlated with the clinical course of the disease.

While GI GVHD is a common complication following HCT there remain many barriers to its consistent and accurate diagnosis. First, diagnosis is dependent on appropriate tissue sampling. Visible lesions are frequently absent19 and endoscopic findings can be diffuse and nonspecific. There is also no consensus on the optimal location of the GI tract for biopsies or number of biopsies needed to secure a diagnosis. There is also frequent discordance between biopsy specimens from the upper and lower GI tract20. Second, patients presenting early in the course of GVHD may have subtle histopathological findings that may be missed or not yet present. At the onset of GVHD few apoptotic cells may be seen and crypt loss and mucosal damage may yet to have occured18. Lastly, there are also confounding factors that can lead to the misdiagnosis of GVHD that inc

• Perform longitudinal immune analysis on T cells and B cells purified from patients with allo- and auto-immune diseases and those receiving cellular immunotherapies. [Срок оценки: 1 year]
• Perform microbiome analysis longitudinally in patients with auto- and allo-immune diseases. [Срок оценки: 1 year]
• Perform longitudinal immune analysis on T- and B-cells as well as measurements of serum antibody titers from patients with allo- and auto-immune disorders who receive immunization against COVID-19. [Срок оценки: 1 year]