Cotargeting EBV lytic as well as latent cycle antigens increases T-cell potency against lymphoma.

ABSTRACT: The remarkable efficacy of Epstein-Barr virus (EBV)-specific T cells for the treatment of posttransplant lymphomas has not been reproduced for EBV-positive (EBV+) malignancies outside the transplant setting. This is because of, in part, the heterogeneous expression and poor immunogenicity of the viral antigens expressed, namely latent membrane proteins 1 and 2, EBV nuclear antigen 1, and BamHI A rightward reading frame 1 (type-2 [T2] latency). However, EBV lytic cycle proteins are also expressed in certain EBV+ malignancies and, because several EBV lytic cycle proteins are abundantly expressed, have oncogenic activity, and likely contribute to malignancy, we sought and identified viral lytic-cycle transcripts in EBV+ Hodgkin lymphoma biopsies. This provided the rationale for broadening the target antigen-specific repertoire of EBV-specific T cells (EBVSTs) for therapy. We stimulated, peripheral blood mononuclear cells from healthy donors and patients with EBV+ lymphoma with both lytic and latent cycle proteins to produce broad repertoire (BR) EBVSTs. Compared with T2 antigen-specific EBVSTs, BR-EBVSTs more rapidly cleared autologous EBV+ tumors in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and produced higher levels of proinflammatory cytokines that should reactivate the immunosuppressive tumor microenvironment leading to epitope spreading. Our results confirm that lytic cycle antigens are clinically relevant targets for EBV+ lymphoma and underpin the rationale for integrating BR-EBVSTs as a therapeutic approach for relapsed/refractory EBV+ lymphoma (www.clinicaltrials.gov identifiers: #NCT01555892 and #NCT04664179), as well as for other EBV-associated malignancies.

Genital Tucking Practices in Transgender and Gender Diverse Patients.

Genital tucking (tucking) is the practice of hiding or minimizing the appearance of one's genitals and gonads. We aimed to better understand the prevalence of tucking and its potential effect on behavior and health. An online questionnaire was distributed to adults with a diagnosis of gender dysphoria or gender incongruence (n = 98). The risk of side effects increased with the length of tucking sessions (P = 0.046) with many patients avoiding medical care despite experiencing side effects. Health care providers should empathetically discuss tucking and its potential risks and benefits with transgender and gender diverse patients. Further research is needed to better quantify the potential risks involved with tucking and to assist in developing educational resources.

Assessing efficacy of a web-based smoking cessation tool - QuitAdvisorMD: Protocol for a practice-based, clustered, randomized control trial.

Background: Smoking remains the leading cause of preventable death, yet physicians inconsistently provide best-practices cessation advice to smokers. Point-of-care digital health tools can prompt and assist physicians to provide improved smoking cessation counseling. QuitAdvisorMD is a comprehensive web-based counseling and management digital health tool designed to guide smoking cessation counseling at the point-of-care. The tool enables clinicians to assess patient readiness to change and then deliver stage-appropriate interventions, while also incorporating Motivational Interviewing techniques. We present the research protocol to assess the efficacy of QuitAdvisorMD to change frequency and quality of smoking cessation counseling and its effect on patient quit rates. Methods: A practice-based, clustered, randomized controlled trial will be used to evaluate QuitAdvisorMD. Cluster design will be used where patients are clustered within primary care practices and practices will be randomized to either the intervention (QuitAdvisorMD) or control group. The primary outcome is frequency and quality of clinician initiated smoking cessation counseling. Secondary outcomes include, 1) changes in physician knowledge, skills and perceived self-efficacy in providing appropriate stage-based smoking cessation counseling and 2) patient quit attempts. Analyses will be conducted to determine pre- and post-test individual clinician outcomes and between intervention and control group practices for patient outcomes. Conclusion: Results from this study will provide important insights regarding the ability of an integrated, web-based counseling and management tool (QuitAdvisorMD) to impact both the quality and efficacy of smoking cessation counseling in primary care settings.

Constitutive Interleukin-7 Cytokine Signaling Enhances the Persistence of Epstein-Barr Virus-Specific T-Cells.

The efficacy of therapeutic T-cells is limited by a lack of positive signals and excess inhibitory signaling in tumor microenvironments. We previously showed that a constitutively active IL7 receptor (C7R) enhanced the persistence, expansion, and anti-tumor activity of T-cells expressing chimeric antigen receptors (CARs), and C7R-modified GD2.CAR T-cells are currently undergoing clinical trials. To determine if the C7R could also enhance the activity of T-cells recognizing tumors via their native T-cell receptors (TCRs), we evaluated its effects in Epstein-Barr virus (EBV)-specific T-cells (EBVSTs) that have produced clinical benefits in patients with EBV-associated malignancies. EBVSTs were generated by stimulation of peripheral blood T-cells with overlapping peptide libraries spanning the EBV lymphoma antigens, LMP1, LMP2, and EBNA 1, followed by retroviral vector transduction to express the C7R. The C7R increased STAT5 signaling in EBVSTs and enhanced their expansion over 30 days of culture in the presence or absence of exogenous cytokines. C7R-EBVSTs maintained EBV antigen specificity but were dependent on TCR stimulation for continued expansion. C7R-EBVSTs produced more rapid lymphoma control in a murine xenograft model than unmodified EBVSTs and persisted for longer. The findings have led to a clinical trial, evaluating C7R-EBVSTs for the treatment of refractory or relapsed EBV-positive lymphoma (NCT04664179).

Structural surfaceomics reveals an AML-specific conformation of integrin ß2 as a CAR T cellular therapy target.

Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here we explore the hypothesis that tumor cells express cancer-specific surface protein conformations that are invisible to standard target discovery pipelines evaluating gene or protein expression, and these conformations can be identified and immunotherapeutically targeted. We term this strategy integrating cross-linking mass spectrometry with glycoprotein surface capture 'structural surfaceomics'. As a proof of principle, we apply this technology to acute myeloid leukemia (AML), a hematologic malignancy with dismal outcomes and no known optimal immunotherapy target. We identify the activated conformation of integrin ß2 as a structurally defined, widely expressed AML-specific target. We develop and characterize recombinant antibodies to this protein conformation and show that chimeric antigen receptor T cells eliminate AML cells and patient-derived xenografts without notable toxicity toward normal hematopoietic cells. Our findings validate an AML conformation-specific target antigen and demonstrate a tool kit for applying these strategies more broadly.

Engaging Family Medicine Residents in a Structured Patient Panel Reassignment Process.

BACKGROUND AND OBJECTIVES: The patient panels of graduating residents must be reassigned by the end of residency. This process affects over 1 million patients annually within the specialty of family medicine. The purpose of this project was to implement a structured, year-end reassignment system in a family medicine residency program. METHODS: Our structured reassignment process took place from December 2017 through June 2020. Panel lists of current, active patients were generated and residents were responsible for reassigning their own panels during a panel reassignment night. We created a tip sheet that addressed patient complexity and continuity, a risk stratification algorithm based on patients' medical and social complexity, and a tool that tracked the number of patients assigned to each future provider. Outcome measures included a resident satisfaction survey administered in 2018-2020 and patient-provider continuity measured with a run chart from December 2016 through August 2020. RESULTS: The resident survey response rate was 75%. Seventy-three percent felt the panel reassignment night was very helpful; 87% thought the reassignment timeline was extremely reasonable, and 87% indicated that they had the necessary information to reassign their patients. Residents also felt confident that their patients were reassigned appropriately (33% extremely confident, 67% somewhat confident). Patient continuity improved with a 13-point run above the median, indicating nonrandom variation. Patient continuity remained above the median until the impact of COVID-19 in April 2020. CONCLUSION: Our structured reassignment process was received positively by residents and resulted in improved patient continuity.

Enhancing your scholarship as a family medicine junior faculty member.

Family medicine faculty are often expected to produce some form of scholarship as members of academic departments. However, this can be challenging given a range of contextual factors, including limited research capacity in many departments, increased competition for funding and individual challenges around balancing multiple roles, unclear expectations and lack of mentorship, to name a few. The purpose of this reflection is to discuss seven content areas that might be addressed by faculty in order to promote scholarship, particularly among junior faculty. These include: 1) knowing your academic track and its associated expectations by rank, as well as the scholarship expectations within your department; 2) considering your personal goals, interests, professional development needs and the relationship between meaningful work and burnout; 3) starting small and building towards a niche content area; 4) finding collaborators and the benefits of collaboration; 5) seeking alignment between your scholarship and work that you already are performing; 6) educating yourself about available outlets for scholarship and 7) seeking mentorship.

Utilizing the F3App to Capture Learner Feedback About Faculty Teaching.

BACKGROUND AND OBJECTIVES: The Accreditation Council for Graduate Medical Education requires soliciting learner feedback on faculty teaching, although gathering meaningful feedback is challenging in the medical education environment. We developed the Faculty Feedback Facilitator (F3App), a mobile application that allows for real-time capture of narrative feedback by residents. The purpose of our study was to assess efficacy, usability, and acceptability of the F3App in family medicine residency programs. METHODS: Residents, faculty, and program directors (PDs) from eight residency programs participated in a beta test of the F3App from November 2017 to May 2018; participants completed pre- and postimplementation surveys about their evaluation process and the F3App. We interviewed PDs, and analyzed responses using a thematic analysis approach. RESULTS: Survey results showed significant postimplementation increases in faculty agreement that accessing evaluations is easy (42%), evaluations are an effective way to communicate feedback (34%), feedback is actionable and meaningful (24%), and the current system provides meaningful data for promotion (33%). Among residents, agreement that the current system allows meaningful information sharing and is easy to use increased significantly, by 17% each. The proportion of residents agreeing they were comfortable providing constructive criticism increased significantly (22%). PDs generally reported that residents were receptive to using the F3App, found it quick and easy to use, and that feedback provided was meaningful. CONCLUSIONS: Participating programs reviewed the F3App positively as a tool to gather narrative feedback from learners on faculty teaching.

Fat-Phobic and Non-Fat-Phobic Anorexia Nervosa: A Conjoint Analysis on the Importance of Shape and Weight.

With the introduction of new diagnostic criteria in DSM-5, fear of weight gain no longer represents a sine qua non-criterion for the diagnosis of anorexia nervosa (AN). This is of relevance as a subgroup of individuals with AN denies fear of weight gain as the reason for restrictive eating but still remain at a very low weight. As self-reports are susceptible to bias, other methods are needed to confirm the existence of the subtype in order to provide adapted treatment. Therefore, we aimed to measure fear of weight gain using a novel method in clinical psychology, the conjoint analysis (CA). Relative importance and preference scores for various life aspects, including appearance/shape and weight were assessed in women with fat-phobic AN (FP-AN, n = 30), NFP-AN (n = 7), and healthy controls (n = 29). Individuals with FP-AN showed a significant lower preference for weight gain versus weight maintenance than HC (p = 0.011, η p 2 = 0.107). Correlation between explicitly assessed drive for thinness and CA score was low. As expected, in FP-AN the explicitly endorsed fear of weight gain was confirmed by the marked preference for weight maintenance compared to HC, while for NFP-AN explicit and implicit measures diverged, indicating that against their self-report they may experience at least some fear of weight gain. The utility of CA as a tool to measure fear of weight gain - and potentially other psychopathological constructs -requires further confirmation.

Common Medical Concerns of the Female Athlete.

Women are increasingly participating in more and more sporting activities. For years, women athletes have been treated as the "female" equivalent of male athletes, with similar medical approaches but this is changing. The concept that women are unique in their "athletic arena" is further underscored with emerging scientific evidence--from the physiologic details not visible to the eye, to the more overt biomechanical and anatomic differences. We review a handful of conditions active women potentially may encounter: pregnancy, the female athlete triad, patellofemoral pain, potential injuries to the anterior cruciate ligament, and anemia.

T-Cell Receptor Stimulation Enhances the Expansion and Function of CD19 Chimeric Antigen Receptor-Expressing T Cells.

PURPOSE: Current protocols for CD19 chimeric antigen receptor-expressing T cells (CD19.CAR-T cells) require recipients to tolerate preinfusion cytoreductive chemotherapy, and the presence of sufficient target antigen on normal or malignant B cells. PATIENTS AND METHODS: We investigated whether additional stimulation of CD19.CAR-T cells through their native receptors can substitute for cytoreductive chemotherapy, inducing expansion and functional persistence of CD19.CAR-T even in patients in remission of B-cell acute lymphocytic leukemia. We infused a low dose of CD19.CAR-modified virus-specific T cells (CD19.CAR-VST) without prior cytoreductive chemotherapy into 8 patients after allogeneic stem cell transplant. RESULTS: Absent virus reactivation, we saw no CD19.CAR-VST expansion. In contrast, in patients with viral reactivation, up to 30,000-fold expansion of CD19.CAR-VSTs was observed, with depletion of CD19+ B cells. Five patients remain in remission at 42-60+ months. CONCLUSIONS: Dual T-cell receptor and CAR stimulation can thus potentiate effector cell expansion and CAR-target cell killing, even when infusing low numbers of effector cells without cytoreduction.

Residency Faculty Teaching Evaluation: What Do Faculty, Residents, and Program Directors Want?

BACKGROUND AND OBJECTIVES: The Accreditation Council for Graduate Medical Education Common Residency Program Requirements stipulate that each faculty member's performance be evaluated annually. Feedback is essential to this process, yet the culture of medicine poses challenges to developing effective feedback systems. The current study explores existing and ideal characteristics of faculty teaching evaluation systems from the perspectives of key stakeholders: faculty, residents, and residency program directors (PDs). METHODS: We utilized two qualitative approaches: (1) confidential semistructured telephone interviews with PDs from a convenience sample of eight family medicine residency programs, (2) qualitative responses from an anonymous online survey of faculty and residents in the same eight programs. We used inductive thematic analysis to analyze the interviews and survey responses. Data collection occurred in the fall of 2017. RESULTS: All eight (100%) of the PDs completed interviews. Survey response rates for faculty and residents were 79% (99/126) and 70% (152/216), respectively. Both PD and faculty responses identified a desire for actionable, real-time, frequent feedback used to foster continued professional development. Themes unique to faculty included easy accessibility and feedback from peers. Residents expressed an interest in in-person feedback and a process minimizing potential retribution. Residents indicated that feedback should be based on shared understanding of what skill(s) the faculty member is trying to address. CONCLUSIONS: PDs, faculty, and residents share a desire to provide faculty with meaningful, specific, and real-time feedback. Programs should strive to provide a culture in which feedback is an integral part of the learning process for both residents and faculty.

School Absenteeism in Children and Adolescents.

Frequent school absenteeism has immediate and long-term negative effects on academic performance, social functioning, high school and college graduation rates, adult income, health, and life expectancy. Previous research focused on distinguishing between truancy and anxiety-driven school refusal, but current policy has shifted to reducing absenteeism for any reason. Chronic absenteeism appears to be driven by overlapping medical, individual, family, and social factors, including chronic illness, mental health conditions, bullying, perceived lack of safety, health problems or needs of other family members, inconsistent parenting, poor school climate, economic disadvantage, and unreliable transportation. Family physicians are well positioned to identify patients with frequent absences, intervene early, and tailor treatment plans to the patient's medical and social needs. Informing parents of the link between school attendance and achievement can be effective in reducing absences. If absenteeism is caused by chronic illness, management should include clear expectations about school attendance and care coordination with school personnel. Mental health conditions that interfere with school attendance can often be treated with cognitive behavior therapy and/or pharmacotherapy. When assessing a child with frequent absences, physicians should inquire about bullying, even if the patient is not known to identify with a vulnerable group. Families and schools are key collaborators in interventions via parent education, parental mental health treatment, and school-based intervention programs.

Efficacy of intracellular immune checkpoint-silenced DC vaccine.

BACKGROUND: DC-based tumor vaccines have had limited clinical success thus far. SOCS1, a key inhibitor of inflammatory cytokine signaling, is an immune checkpoint regulator that limits DC immunopotency. METHODS: We generated a genetically modified DC (gmDC) vaccine to perform immunotherapy. The adenovirus (Ad-siSSF) delivers two tumor-associated antigens (TAAs), survivin and MUC1; secretory bacterial flagellin for DC maturation; and an RNA interference moiety to suppress SOCS1. A 2-stage phase I trial was performed for patients with relapsed acute leukemia after allogenic hematopoietic stem cell transplantation: in stage 1, we compared the safety and efficacy between gmDC treatment (23 patients) and standard donor lymphocyte infusion (25 patients); in stage 2, we tested the efficacy of the gmDC vaccine for 12 acute myeloid leukemia (AML) patients with early molecular relapse. RESULTS: gmDCs elicited potent TAA-specific CTL responses in vitro, and the immunostimulatory activity of gmDC vaccination was demonstrated in rhesus monkeys. A stage 1 study established that this combinatory gmDC vaccine is safe in acute leukemia patients and yielded improved survival rate. In stage 2, we observed a complete remission rate of 83% in 12 relapsed AML patients. Overall, no grade 3 or grade 4 graft-versus-host disease incidence was detected in any of the 35 patients enrolled. CONCLUSIONS: This study, with combinatory modifications in DCs, demonstrates the safety and efficacy of SOCS1-silenced DCs in treating relapsed acute leukemia. TRIAL REGISTRATION: ClinicalTrials.gov NCT01956630. FUNDING: National Institute of Health (R01CA90427); the Key New Drug Development and Manufacturing Program of the "Twelfth Five-Year Plan" of China (2011ZX09102-001-29); and Clinical Application Research of Beijing (Z131107002213148).

Celiac Disease and Gluten Sensitivity.

Celiac disease is an immune-mediated enteropathy triggered by gluten that affects genetically predisposed individuals, typically causing intestinal symptoms and malabsorption. Diagnosis requires stepwise evaluation with anti-tissue transglutaminase IgA and histologic analysis of the small bowel. Strict adherence to a gluten-free diet is the primary treatment. Patients with symptoms thought to be related to gluten but without evidence of celiac disease are difficult to diagnose and treat. Consider first advising general nutritional improvements. If symptoms persist, involve a trained dietitian for restrictive diets and consider evaluation for small intestinal bacterial overgrowth or other treatments for irritable bowel syndrome.

Comprehensive Approach for Identifying the T Cell Subset Origin of CD3 and CD28 Antibody-Activated Chimeric Antigen Receptor-Modified T Cells.

The outcome of therapy with chimeric Ag receptor (CAR)-modified T cells is strongly influenced by the subset origin of the infused T cells. However, because polyclonally activated T cells acquire a largely CD45RO+CCR7- effector memory phenotype after expansion, regardless of subset origin, it is impossible to know which subsets contribute to the final T cell product. To determine the contribution of naive T cell, memory stem T cell, central memory T cell, effector memory T cell, and terminally differentiated effector T cell populations to the CD3 and CD28-activated CAR-modified T cells that we use for therapy, we followed the fate and function of individually sorted CAR-modified T cell subsets after activation with CD3 and CD28 Abs (CD3/28), transduction and culture alone, or after reconstitution into the relevant subset-depleted population. We show that all subsets are sensitive to CAR transduction, and each developed a distinct T cell functional profile during culture. Naive-derived T cells showed the greatest rate of proliferation but had more limited effector functions and reduced killing compared with memory-derived populations. When cultured in the presence of memory T cells, naive-derived T cells show increased differentiation, reduced effector cytokine production, and a reduced reproliferative response to CAR stimulation. CD3/28-activated T cells expanded in IL-7 and IL-15 produced greater expansion of memory stem T cells and central memory T cell-derived T cells compared with IL-2. Our strategy provides a powerful tool to elucidate the characteristics of CAR-modified T cells, regardless of the protocol used for expansion, reveals the functional properties of each expanded T cell subset, and paves the way for a more detailed evaluation of the effects of manufacturing changes on the subset contribution to in vitro-expanded T cells.

Large-Scale Culture and Genetic Modification of Human Natural Killer Cells for Cellular Therapy.

Recent advances in methods for the ex vivo expansion of human natural killer (NK) cells have facilitated the use of these powerful immune cells in clinical protocols. Further, the ability to genetically modify primary human NK cells following rapid expansion allows targeting and enhancement of their immune function. We have successfully adapted an expansion method for primary NK cells from peripheral blood mononuclear cells or from apheresis products in gas permeable rapid expansion devices (G-Rexes). Here, we describe an optimized protocol for rapid and robust NK cell expansion as well as a method for highly efficient retroviral transduction of these ex vivo expanded cells. These methodologies are good manufacturing practice (GMP) compliant and could be used for clinical-grade product manufacturing.

Early transduction produces highly functional chimeric antigen receptor-modified virus-specific T-cells with central memory markers: a Production Assistant for Cell Therapy (PACT) translational application.

BACKGROUND: Virus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients, eliminate virus infections, then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs), they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs, but there was little apparent expansion of these cells in patients. In that study, VSTs were gene-modified on day 19 of culture and we hypothesized that by this time, sufficient T-cell differentiation may have occurred to limit the subsequent proliferative capacity of the transduced T-cells. To facilitate the clinical testing of this hypothesis in a project supported by the NHLBI-PACT mechanism, we developed and optimized a good manufacturing practices (GMP) compliant method for the early transduction of VSTs directed to Epstein-Barr virus (EBV), Adenovirus (AdV) and cytomegalovirus (CMV) using a CAR directed to the tumor-associated antigen disialoganglioside (GD2). RESULTS: Ad-CMVpp65-transduced EBV-LCLs effectively stimulated VSTs directed to all three viruses (triVSTs). Transduction efficiency on day three was increased in the presence of cytokines and high-speed centrifugation of retroviral supernatant onto retronectin-coated plates, so that under optimal conditions up to 88% of tetramer-positive VSTs expressed the GD2.CAR. The average transduction efficiency of early-and late transduced VSTs was 55 ± 4% and 22 ± 5% respectively, and early-transduced VSTs maintained higher frequencies of T cells with central memory or intermediate memory phenotypes. Early-transduced VSTs also had higher proliferative capacity and produced higher levels of TH1 cytokines IL-2, TNF-α, IFN-γ, MIP-1α, MIP-1ß and other cytokines in vitro. CONCLUSIONS: We developed a rapid and GMP compliant method for the early transduction of multivirus-specific T-cells that allowed stable expression of high levels of a tumor directed CAR. Since a proportion of early-transduced CAR-VSTs had a central memory phenotype, they should expand and persist in vivo, simultaneously protecting against infection and targeting residual malignancy. This manufacturing strategy is currently under clinical investigation in patients receiving allogeneic HSCT for relapsed neuroblastoma and B-cell malignancies (NCT01460901 using a GD2.CAR and NCT00840853 using a CD19.CAR).