Redefining our vision: an updated guide to the ocular immune system.

Balanced immune responses in the eyes are crucial to preserve vision. The ocular immune system has long been considered distinct, owing to the so-called 'immune privilege' of its component tissues. More recently, intravital imaging and transcriptomic techniques have reshaped scientific understanding of the ocular immune landscape, such as revealing the specialization of immune cell populations in the various tissues of the eye. As knowledge of the phenotypes of corneal and retinal immune cells has evolved, links to both the systemic immune system, and the central and peripheral nervous systems, have been identified. Using intravital imaging, T cells have recently been found to reside in, and actively patrol, the healthy human cornea. Disease-associated retinal microglia with links to retinal degeneration have also been identified. This Review provides an updated guide to the ocular immune system, highlighting current knowledge of the immune cells that are present in steady-state and specific diseased ocular tissues, as well as evidence for their relationship to systemic disease. In addition, we discuss emerging intravital imaging techniques that can be used to visualize immune cell morphology and dynamics in living human eyes and how these could be applied to advance understanding of the human immune system.

TEMPRO: nanobody melting temperature estimation model using protein embeddings.

Single-domain antibodies (sdAbs) or nanobodies have received widespread attention due to their small size (~ 15 kDa) and diverse applications in bio-derived therapeutics. As many modern biotechnology breakthroughs are applied to antibody engineering and design, nanobody thermostability or melting temperature (Tm) is crucial for their successful utilization. In this study, we present TEMPRO which is a predictive modeling approach for estimating the Tm of nanobodies using computational methods. Our methodology integrates various nanobody biophysical features to include Evolutionary Scale Modeling (ESM) embeddings, NetSurfP3 structural predictions, pLDDT scores per sdAb region from AlphaFold2, and each sequence's physicochemical characteristics. This approach is validated with our combined dataset containing 567 unique sequences with corresponding experimental Tm values from a manually curated internal data and a recently published nanobody database, NbThermo. Our results indicate the efficacy of protein embeddings in reliably predicting the Tm of sdAbs with mean absolute error (MAE) of 4.03 °C and root mean squared error (RMSE) of 5.66 °C, thus offering a valuable tool for the optimization of nanobodies for various biomedical and therapeutic applications. Moreover, we have validated the models' performance using experimentally determined Tms from nanobodies not found in NbThermo. This predictive model not only enhances nanobody thermostability prediction, but also provides a useful perspective of using embeddings as a tool for facilitating a broader applicability of downstream protein analyses.

Engineering Outer Membrane Vesicles to Carry Enzymes: Encapsulation, Isolation, Characterization, and Modification.

Outer membrane vesicles (OMVs) are small, spherical, nanoscale proteoliposomes released from Gram-negative bacteria that play an important role in cellular defense, pathogenesis, and signaling, among other functions. The functionality of OMVs can be enhanced by engineering developed for biomedical and biochemical applications. Here, we describe methods for directed packaging of enzymes into bacterial OMVs of E. coli using engineered molecular systems, such as localizing proteins to the inner or outer surface of the vesicle. Additionally, we detail some modification strategies for OMVs such as lyophilization and surfactant conjugation that enable the protection of activity of the packaged enzyme when exposed to non-physiological conditions such as elevated temperature, organic solvents, and repeated freeze/thaw that otherwise lead to a substantial loss in the activity of the free enzyme.

Neuro-Ophthalmic Manifestations of Adult Polyglucosan Body Disease.

BACKGROUND: Adult polyglucosan body disease (APBD) is caused by a deficiency in glycogen branching enzyme that leads to polyglucosan accumulation in multiple organs. It has a progressive clinical course with prominent neurologic manifestations. We aim to describe the neuro-ophthalmic manifestations of APBD. METHODS: This is a case series of 3 individuals with genetically proven APBD. Written informed consent was provided by the brothers. We also performed a literature review on the current state of knowledge on APBD through PubMed. RESULTS: Brother 1 developed gait imbalance and length-dependent polyneuropathy in his 40s followed by progressive urinary symptoms in his 50s. He reported diplopia and blurry vision in his 60s. Neuro-ophthalmic assessment revealed bilateral optic neuropathy, convergence insufficiency, and a right fourth nerve palsy. Genetic testing showed a homozygous pathogenic variant in GBE1 c.986A>C p.Tyr329Ser. Brother 2 developed progressive urinary symptoms in his 40s that were followed by cognitive deficits, length-dependent polyneuropathy, and lower extremity weakness in his 50s and 60s. He reported blurred vision, and neuro-ophthalmic evaluation revealed bilateral optic neuropathy. Genetic testing revealed the same variant as Brother 1, GBE1 c.986A>C p.Tyr329Ser. Brother 3 developed progressive urinary urgency and lower extremity weakness in his 50s followed by a length-dependent polyneuropathy in his 60s. He reported diplopia and blurry vision in his 70s. Neuro-ophthalmic assessment revealed bilateral optic neuropathy and convergence insufficiency. Genetic testing revealed the same variant as Brothers 1 and 2, GBE1 c.986A>C p.Tyr329Ser. CONCLUSIONS: There is an array of afferent and efferent neuro-ophthalmic manifestations in APBD. Neuro-ophthalmic evaluation is crucial in evaluating and treating patients with APBD, particularly in those with visual dysfunction.

Intranasal Self-Adjuvanted Lipopeptide Vaccines Elicit High Antibody Titers and Strong Cellular Responses against SARS-CoV-2.

Despite concerted efforts to tackle the COVID-19 pandemic, the persistent transmission of SARS-CoV-2 demands continued research into novel vaccination strategies to combat the virus. In light of this, intranasally administered peptide vaccines, particularly those conjugated to an immune adjuvant to afford so-called "self-adjuvanted vaccines", remain underexplored. Here, we describe the synthesis and immunological evaluation of self-adjuvanting peptide vaccines derived from epitopes of the spike glycoprotein of SARS-CoV-2 covalently fused to the potent adjuvant, Pam2Cys, that targets toll-like receptor 2 (TLR2). When administered intranasally, these vaccines elicited a strong antigen-specific CD4+ and CD8+ T-cell response in the lungs as well as high titers of IgG and IgA specific to the native spike protein of SARS-CoV-2. Unfortunately, serum and lung fluid from mice immunized with these vaccines failed to inhibit viral entry in spike-expressing pseudovirus assays. Following this, we designed and synthesized fusion vaccines composed of the T-cell epitope discovered in this work, covalently fused to epitopes of the receptor-binding domain of the spike protein reported to be neutralizing. While antibodies elicited against these fusion vaccines were not neutralizing, the T-cell epitope retained its ability to stimulate strong antigen-specific CD4+ lymphocyte responses within the lungs. Given the Spike(883-909) region is still completely conserved in SARS-CoV-2 variants of concern and variants of interest, we envision the self-adjuvanting vaccine platform reported here may inform future vaccine efforts.

Towards targeting the breast cancer immune microenvironment.

The tumour immune microenvironment is shaped by the crosstalk between cancer cells, immune cells, fibroblasts, endothelial cells and other stromal components. Although the immune tumour microenvironment (TME) serves as a source of therapeutic targets, it is also considered a friend or foe to tumour-directed therapies. This is readily illustrated by the importance of T cells in triple-negative breast cancer (TNBC), culminating in the advent of immune checkpoint therapy in combination with cytotoxic chemotherapy as standard of care for both early and advanced-stage TNBC, as well as recent promising signs of efficacy in a subset of hormone receptor-positive disease. In this Review, we discuss the various components of the immune TME in breast cancer and therapies that target or impact the immune TME, as well as the complexity of host physiology.

Does Preservation of Metacarpophalangeal Joint Vascularity Protect Against Physeal Arrest in Index Finger Pollicization?

PURPOSE: To determine whether preservation of blood supply to the index metacarpophalangeal joint decreases the rate of physeal arrest. METHODS: A retrospective review of 41 pollicized digits in 35 patients with 2-year minimum radiographic follow-up was conducted at a single institution. Other complications evaluated included nonunion at the pollicized digit base and clinical instability at the new carpometacarpal joint. Findings were compared to historical controls, which were performed by our group prior to routine identification and sparing of the metacarpophalangeal joint blood supply. No other modifications to surgical technique were made between the previous and current patient cohorts. RESULTS: Two pollicized digits in two different patients had radiographic evidence of physeal arrest, one of which was partial and the other complete, for an arrest rate of 4.9%. This was significantly less than the arrest rate in our historical cohort of 24.7% (21 of 85 patients). Five patients did not have radiographic bony union at the base of the index metacarpal, but only one patient had clinical instability at the new carpometacarpal joint. CONCLUSIONS: Significantly fewer patients who underwent index finger pollicization with preservation of the metacarpophalangeal joint blood supply went on to develop physeal arrest when compared to patients who underwent pollicization prior to adoption of this technique. This finding suggests that sparing of the physeal blood supply is preventative against proximal phalanx physeal arrest. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

MICK (Mobile Integrated Cognitive Kit) App for Concussion Assessment in a Youth Ice Hockey League.

BACKGROUND: Visual symptoms are common after concussion. Rapid automatized naming (RAN) tasks are simple performance measures that demonstrate worse time scores in the setting of acute or more remote injury. METHODS: We evaluated the capacity for the Mobile Universal Lexicon Evaluation System (MULES) and Staggered Uneven Number (SUN) testing to be feasibly administered during preseason testing in a cohort of youth ice hockey athletes using a novel computerized app, the Mobile Integrated Cognitive Kit (MICK). Participants from a youth hockey league underwent preseason testing. RESULTS: Among 60 participants, the median age was 13 years (range 6-17). The median best time for the MULES was 49.8 seconds (range = 34.2-141.0) and the median best time for the SUN was 70.1 (range = 36.6-200.0). As is characteristic of timed performance measures, there were learning effects between the first and second trials for both the MULES (median improvement = 10.6 seconds, range = -32.3 to 92.0, P < 0.001, Wilcoxon signed-rank test) and SUN (median improvement = 2.4 seconds, range= -8.0 to 15.1, P = 0.001, Wilcoxon signed-rank test). Age was a predictor of best baseline times, with longer (worse) times for younger participants for MULES (P < 0.001, rs = -0.67) and SUN (P < 0.001, rs = -0.54 Spearman rank correlation). Degrees of learning effect did not vary with age (P > 0.05, rs = -0.2). CONCLUSIONS: Vision-based RAN tasks, such as the MULES and SUN, can be feasibly administered using the MICK app during preseason baseline testing in youth sports teams. The results suggest that more frequent baseline tests are necessary for preadolescent athletes because of the relation of RAN task performance to age.

AI in Neuro-Ophthalmology: Current Practice and Future Opportunities.

BACKGROUND: Neuro-ophthalmology frequently requires a complex and multi-faceted clinical assessment supported by sophisticated imaging techniques in order to assess disease status. The current approach to diagnosis requires substantial expertise and time. The emergence of AI has brought forth innovative solutions to streamline and enhance this diagnostic process, which is especially valuable given the shortage of neuro-ophthalmologists. Machine learning algorithms, in particular, have demonstrated significant potential in interpreting imaging data, identifying subtle patterns, and aiding clinicians in making more accurate and timely diagnosis while also supplementing nonspecialist evaluations of neuro-ophthalmic disease. EVIDENCE ACQUISITION: Electronic searches of published literature were conducted using PubMed and Google Scholar. A comprehensive search of the following terms was conducted within the Journal of Neuro-Ophthalmology: AI, artificial intelligence, machine learning, deep learning, natural language processing, computer vision, large language models, and generative AI. RESULTS: This review aims to provide a comprehensive overview of the evolving landscape of AI applications in neuro-ophthalmology. It will delve into the diverse applications of AI, optical coherence tomography (OCT), and fundus photography to the development of predictive models for disease progression. Additionally, the review will explore the integration of generative AI into neuro-ophthalmic education and clinical practice. CONCLUSIONS: We review the current state of AI in neuro-ophthalmology and its potentially transformative impact. The inclusion of AI in neuro-ophthalmic practice and research not only holds promise for improving diagnostic accuracy but also opens avenues for novel therapeutic interventions. We emphasize its potential to improve access to scarce subspecialty resources while examining the current challenges associated with the integration of AI into clinical practice and research.

Contextual Triggers and Tic Severity Across Life Periods: A Retrospective Analysis in Adults with Tic Disorders.

In tic disorders (TD), tic expression varies across the lifespan and as a function of contextual factors. This study explored connections between tic expression and contextual triggers across life periods in 74 adults (Mage = 23.2) with TDs. The Tic History and Coping Strategies form assessed retrospective self-reports of contextual antecedents, consequences, and tic severity during four life periods (middle school; 9th/10th grade; 11th/12th grade; college/work) and past month. Tics reportedly worsened during and after school in school-aged years and worsened in the evening during college/work years. Stress and anxiety were reported to consistently trigger tics across time. The impact of activities, places, and emotions did not differ across life periods. Attention-based consequences, most prevalent during middle school, were more common than escape- or avoidance-related consequences across all periods. Findings illuminate how contextual factors may influence tics across life periods and underscore the consistent impact of tic-triggering emotions and attention-related consequences.

Phase II Trial of Afatinib in Patients With EGFR-Mutated Solid Tumors Excluding Lung Cancer: Results From NCI-MATCH ECOG-ACRIN Trial (EAY131) Subprotocol A.

PURPOSE: National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) was a multicohort phase 2 trial that assigned patients with advanced pretreated cancers to molecularly targeted therapies on the basis of tumor genomic testing. NCI-MATCH Arm A evaluated afatinib, an EGFR tyrosine kinase inhibitor (TKI) approved for advanced non-small cell lung cancer, in patients with tumors other than lung cancer harboring EGFR mutations. METHODS: Patients with advanced pretreated cancers other than lung cancer found to have selected actionable EGFR mutations were offered participation in Arm A. Previous therapy with an EGFR TKI was not allowed. Patients received afatinib 40 mg once daily continuously until disease progression or unacceptable toxicity. The primary end point was objective response rate (ORR). Secondary end points included progression-free survival (PFS), 6-month PFS, and overall survival (OS). RESULTS: Seventeen patients received protocol therapy. Tumor types included glioblastoma multiforme (GBM) (13), gliosarcoma (1), adenocarcinoma not otherwise specified (NOS) (2), and adenosquamous carcinoma of the breast (1). Fifty-nine percent of patients received ≥2 lines of previous therapy. The ORR was 11.8% (90% CI, 2.1 to 32.6), with one complete response lasting 16.4 months (GBM harboring a rare exon 18 EGFR-SEPT14 fusion) and one partial response lasting 12.8 months (adenocarcinoma NOS with the classic EGFR mutation, p.Glu746_Ala750del). Three patients had stable disease. The 6-month PFS was 15% (90% CI, 0 to 30.7); the median OS was 9 months (90% CI, 4.6 to 14.0). Rash and diarrhea were the most common toxicities. CONCLUSION: Afatinib had modest activity in a cohort of patients with heavily pretreated cancer with advanced nonlung, EGFR-mutated tumors, but the trial's primary end point was not met. Further evaluation of afatinib in GBM with EGFR exon 18 fusions may be of interest.

Admission screening testing of patients and staff N95 respirators are cost-effective in reducing COVID-19 hospital-acquired infections.

BACKGROUND: Coronavirus disease 2019 (COVID-19) outbreaks in acute care settings can have severe consequences for patients due to their underlying vulnerabilities, and can be costly due to additional patient bed-days and the need to replace isolating staff. This study assessed the cost-effectiveness of clinical staff N95 respirators and admission screening testing of patients to reduce COVID-19 hospital-acquired infections. METHODS: An agent-based model was calibrated to data on 178 outbreaks in acute care settings in Victoria, Australia between October 2021 and July 2023. Outbreaks were simulated under different combinations of staff masking (surgical, N95) and patient admission screening testing [none, rapid antigen test (RAT), polymerase chain reaction]. For each scenario, average diagnoses, COVID-19 deaths, quality-adjusted life years from discharged patients, and costs (masks, testing, patient COVID-19 bed-days, staff replacement costs while isolating) from acute COVID-19 were estimated over a 12-month period. FINDINGS: Compared with no admission screening testing and staff surgical masks, all scenarios were cost saving with health gains. Staff N95 respirators + RAT admission screening of patients was the cheapest scenario, saving A$78.4M [95% uncertainty interval (UI) 44.4M-135.3M] and preventing 1543 (95% UI 1070-2146) deaths state-wide per annum. Both interventions were individually beneficial: staff N95 respirators saved A$54.7M and 854 deaths state-wide per annum, while RAT admission screening of patients saved A$57.6M and 1176 deaths state-wide per annum. INTERPRETATION: In acute care settings, staff N95 respirators and admission screening testing of patients can reduce hospital-acquired COVID-19 and COVID-19 deaths, and are cost saving because of reduced patient bed-days and staff replacement needs.

Development of a novel precision applicator for spot treatment of granular agrochemical in wild blueberry.

While spot spraying has gained increasing popularity in recent years, spot application of granule agrochemical has seen little development. Despite the potential for the technology, there currently exists no commercially available granular applicators capable of spot application. Therefore, the goal of this study was to design, build, and lab evaluate a precision applicator for spot applying granular agrochemical in wild blueberry. The design incorporated a John Deere RC2000 with a custom control box, recirculation system, and electrically actuated valves. All components were modified to fit a Valmar 1255 Twin-Roller. The system receives inputs from a predeveloped prescription map and can actuate each of the twelve valves separately to provide individual orifice control. Casoron® G4 was used as the testing agrochemical and in cycling the product pneumatically for 1 hour incurred no significant product degradation (p = 0.110). In lab evaluations, the applicator encountered zero errors in reading prescription maps and actuating the correct valves accordingly. Further, the granule recycling system had zero instances where product built up in the lines or jammed the valves. In all, this project represents the first successful development of a precision granular spot applicator for any cropping system.

Cognitive Disengagement Syndrome in Young Autistic Children, Children with ADHD, and Autistic Children with ADHD.

OBJECTIVE: Cognitive Disengagement Syndrome (CDS; previously called Sluggish Cognitive Tempo) refers to a constellation of cognitive and motor behaviors characterized by a predisposition toward mind wandering (cognitive subdomain) and slowed motor behavior (hypoactive). While there are a number of studies linking CDS traits to greater global impairment in children with attention-deficit/hyperactivity disorder (ADHD) and autistic children, there are few studies examining the prevalence and impact of CDS traits in autistic children with co-occurring ADHD (Autistic+ADHD). The current study explored CDS traits in autistic children with and without co-occurring ADHD, children with ADHD, and neurotypical children. METHODS: Participants were 196 children between 3- and 7-years-of-age comprising four groups: Neurotypical (N = 44), ADHD (N = 51), Autistic (N = 55), and Autistic+ADHD (N = 46). CDS traits, social and communication skills, repetitive behaviors, and sensory processing were all assessed via parent report. RESULTS: Children diagnosed with ADHD, autistic children, and Autistic+ADHD children exhibited similar levels of overall CDS traits. However, when explored separately, Autistic+ADHD children had higher cognitive CDS trait scores compared to children with ADHD alone. Both overall CDS traits and the cognitive subdomain were associated with greater social difficulties, particularly social withdrawal, higher levels of repetitive behaviors, and more sensory sensitivities, regardless of diagnosis. CONCLUSIONS: Findings suggest that CDS traits may be an additional factor directly impact functional outcomes in both autistic and ADHD children. As such, clinicians should be assessing CDS traits in addition to other clinical domains associated with ADHD and autism when developing intervention plans for young neurodiverse children.

Testing the Validity and Reliability of a Standardized Virtual Examination for Concussion.

Background and Objectives: We determined inter-modality (in-person vs telemedicine examination) and inter-rater agreement for telemedicine assessments (2 different examiners) using the Telemedicine Buffalo Concussion Physical Examination (Tele-BCPE), a standardized concussion examination designed for remote use. Methods: Patients referred for an initial evaluation for concussion were invited to participate. Participants had a brief initial assessment by the treating neurologist. After a patient granted informed consent to participate in the study, the treating neurologist obtained a concussion-related history before leaving the examination room. Using the Tele-BCPE, 2 virtual examinations in no specific sequence were then performed from nearby rooms by the treating neurologist and another neurologist. After the 2 telemedicine examinations, the treating physician returned to the examination room to perform the in-person examination. Intraclass correlation coefficients (ICC) determined inter-modality validity (in-person vs remote examination by the same examiner) and inter-rater reliability (between remote examinations done by 2 examiners) of overall scores of the Tele-BCPE within the comparison datasets. Cohen's kappa, κ, measured levels of agreement of dichotomous ratings (abnormality present vs absent) on individual components of the Tele-BCPE to determine inter-modality and inter-rater agreement. Results: For total scores of the Tele-BCPE, both inter-modality agreement (ICC = 0.95 [95% CI 0.86-0.98, p < 0.001]) and inter-rater agreement (ICC = 0.88 [95% CI 0.71-0.95, p < 0.001]) were reliable (ICC >0.70). There was at least substantial inter-modality agreement (κ ≥ 0.61) for 25 of 29 examination elements. For inter-rater agreement (2 telemedicine examinations), there was at least substantial agreement for 8 of 29 examination elements. Discussion: Our study demonstrates that the Tele-BCPE yielded consistent clinical results, whether conducted in-person or virtually by the same examiner, or when performed virtually by 2 different examiners. The Tele-BCPE is a valid indicator of neurologic examination findings as determined by an in-person concussion assessment. The Tele-BCPE may also be performed with excellent levels of reliability by neurologists with different training and backgrounds in the virtual setting. These findings suggest that a combination of in-person and telemedicine modalities, or involvement of 2 telemedicine examiners for the same patient, can provide consistent concussion assessments across the continuum of care.

The Multifaceted Role of Tissue-Resident Memory T Cells.

Regionalized immune surveillance relies on the concerted efforts of diverse memory T cell populations. Of these, tissue-resident memory T (TRM) cells are strategically positioned in barrier tissues, where they enable efficient frontline defense against infections and cancer. However, the long-term persistence of these cells has been implicated in a variety of immune-mediated pathologies. Consequently, modulating TRM cell populations represents an attractive strategy for novel vaccination and therapeutic interventions against tissue-based diseases. Here, we provide an updated overview of TRM cell heterogeneity and function across tissues and disease states. We discuss mechanisms of TRM cell-mediated immune protection and their potential contributions to autoimmune disorders. Finally, we examine how TRM cell responses might be durably boosted or dampened for therapeutic gain.

On second thoughts: Testing the underlying mechanisms of spontaneous future thought.

The human capacity to imagine possible future events unintentionally, with minimal cognitive effort, is termed spontaneous future thought (SFT). This paper addresses an important theoretical question for cognitive science: What are the possible cognitive mechanisms underlying such SFT experiences? We contrasted three hypotheses present in the literature: the online construction hypothesis, the recasting hypothesis, and the memories of future thoughts hypothesis. Study 1 (N = 41) used novel subjective ratings which challenged the recasting mechanism: SFTs were mostly rated as dissimilar to autobiographical memories, suggesting they are not simply past experiences 'recast' as future events. Study 2 (N = 90) used a novel experimental paradigm, comparing effects of voluntary episodic future constructions and non-personal narratives upon subsequent spontaneous thought sampling. Results suggested that voluntary future constructions remain accessible to spontaneous retrieval, supporting the memories of future thoughts hypothesis. This finding, and other data presented across the two studies, still indicates a role for online construction processes in SFT, but further empirical work is needed to clarify how and when constructive processes are engaged in SFT. Taken together, these two studies represent initial efforts to elucidate the mechanisms underlying SFT, providing the first proof-of-principle that deliberately envisioned future events can reappear, without intention, in consciousness at some later time, and further supporting the dual process account of future thinking. These methods and findings provide a firm basis for subsequent experimental and longitudinal research on SFT.

Calcineurin inhibition rescues alloantigen-specific central memory T cell subsets that promote chronic GVHD.

Calcineurin inhibitors (CNIs) constitute the backbone of modern acute graft-versus-host disease (aGVHD) prophylaxis regimens but have limited efficacy in the prevention and treatment of chronic GVHD (cGVHD). We investigated the effect of CNIs on immune tolerance after stem cell transplantation with discovery-based single-cell gene expression and T cell receptor (TCR) assays of clonal immunity in tandem with traditional protein-based approaches and preclinical modeling. While cyclosporin and tacrolimus suppressed the clonal expansion of CD8+ T cells during GVHD, alloreactive CD4+ T cell clusters were preferentially expanded. Moreover, CNIs mediated reversible dose-dependent suppression of T cell activation and all stages of donor T cell exhaustion. Critically, CNIs promoted the expansion of both polyclonal and TCR-specific alloreactive central memory CD4+ T cells (TCM) with high self-renewal capacity that mediated cGVHD following drug withdrawal. In contrast to posttransplant cyclophosphamide (PT-Cy), CSA was ineffective in eliminating IL-17A-secreting alloreactive T cell clones that play an important role in the pathogenesis of cGVHD. Collectively, we have shown that, although CNIs attenuate aGVHD, they paradoxically rescue alloantigen-specific TCM, especially within the CD4+ compartment in lymphoid and GVHD target tissues, thus predisposing patients to cGVHD. These data provide further evidence to caution against CNI-based immune suppression without concurrent approaches that eliminate alloreactive T cell clones.