Alexandre Albanese

Successful hematopoietic cell transplant requires immunosuppression to prevent graft-versus-host disease (GVHD), a lethal, T cell–mediated complication. The phase 3 BMT CTN 1703 trial demonstrated superior GVHD-free/relapse-free survival for posttransplant cyclophosphamide (PT-Cy)–based GVHD prophylaxis versus tacrolimus/methotrexate (Tac/MTX), but the T cell biology underpinning these outcomes remains unknown. To determine this, 324 patients were coenrolled onto BMT CTN 1801 to investigate the distinct T cell immune reconstitution pathways of these regimens, including extensive evaluation of T cell receptor (TCR) repertoire, via sequencing of 2359 longitudinal samples and 180,432,350 T cells. Multimodal T cell analysis revealed that PT-Cy was associated with pan–T cell deletion followed by delayed T cell expansion from a smaller founder population compared with Tac/MTX. This created a T cell diversity bottleneck, leading to reduced TCR repertoire diversity for 2 years posttransplant. Reduced TCR diversity, more common in patients receiving PT-Cy, was linked to protection against chronic GVHD when patients were analyzed without regard to study arm. Leveraging donor- and host-specific TCR clonal analysis, we determined that PT-Cy–mediated T cell depletion also depleted pathogen-specific TCRs, rendering PT-Cy patients more likely to lack detectable graft-derived pathogen-specific TCRs early after transplant, which was associated with an increased risk of severe infection. This study reveals how distinct immunosuppression strategies uniquely remodel T cell immunity and shape divergent transplant outcomes.

Key Points: Pediatric transplant recipients have oligoclonal expansion of CD8 ⁺ T cells and polyclonal expansion of CD4 ⁺ T cells versus healthy children. Analysis of vaccine responses revealed patients with intact T cell immunity but impaired humoral immunity. A subset of pediatric transplant recipients exhibit an atypical B cell population previously associated with excessive immune activation. Background: Immunosuppressive therapy after solid organ transplantation inhibits protective immunity to pathogens and vaccines. However, the specific cell states associated with failure to generate responsiveness to vaccination are not known. Methods: We evaluated a broad spectrum of immune cell states in the peripheral blood of 12 pediatric solid organ transplant recipients (SOTRs) and eight healthy children immediately before receiving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination, by performing single-cell RNA-sequencing, T cell receptor sequencing, and cytometry by time-of-flight. We then evaluated associations between the identified cell states and the development of anti-SARS-CoV-2 S-protein–specific antibody and CD4 ⁺ T cell activation after an initial series of SARS-CoV-2 vaccinations. Results: We found clonal expansion of a subset of CD8 ⁺ effector T cells and a polyclonal expansion of CD4 ⁺ effector T cell populations in SOTRs compared with healthy controls. Responses to vaccination included normal SARS-CoV-2–specific T cell and antibody responses, intact T cell responses but impaired antibody responses, and absence of T cell and antibody responses. Comparison among the prevaccination cell states and responsiveness revealed a higher frequency of atypical B cells in SOTRs with intact T cell but impaired antibody responses compared with healthy controls. Also, T cell-only SOTR responders had lower numbers of immature γ δ T cells compared with SOTR nonresponders. By contrast, there was no definitive cell state that identified an absent immune response in SOTRs, but interactome analysis suggested that robust cellular interactions between myeloid, T cells, and B cells are required for successful responses to vaccination. Conclusions: Our findings in this exploratory observational study suggest that higher frequencies of atypical B cells in the peripheral blood of pediatric SOTRs may identify intact cellular but absent humoral responsiveness to vaccination. Intact T cell responsiveness to antigens may be sufficient to monitor protective immunity after vaccination in SOTRs.

Pediatric Inflammatory Bowel Disease (IBD) remains challenging to treat and difficult to prognosticate. Although multiple immune cell types coordinate pathology in both Crohn’s disease (CD) and ulcerative colitis (UC), specifying which cell types and cell states portend better or worse response to major IBD treatment strategies, including anti-TNF therapies (the only FDA-approved therapy for pediatric IBD), remains challenging. Here, we present the results of the PREDICT study, which enrolled 79 treatment-naïve pediatric patients at the time of diagnostic endoscopy, enabling a comprehensive transcriptomic, histologic, and serologic analysis of 40 CD patients and 16 UC patients, as well as 23 patients with functional gastrointestinal disorders (FGID) who served as pediatric non-inflamed controls. Leveraging these data, we performed a comprehensive analysis of colonic immunology in each of these clinical conditions. Our results indicate that, within the complex landscape of immune pathology in pediatric CD and UC, there is a coordinated shift across the T _H 1-to-T _H 17 immune activation continuum among T cells that is pertinent to anti-TNF response. For CD, this shift defines partial response to anti-TNF treatment. For UC, the landscape is more complex, with both T _H 17 and TFH biology defining disease, and pre-treatment T _H 17 biology contributing to anti-TNF treatment resistance. Related to this, polyreactive TCR phenotypes within UC T _FH cells are correlated with both germinal center activity and the frequency of IgG1 plasma cells, yet opposed to the T _H 17 signatures associated with anti-TNF nonresponse. The elucidation of these distinct mechanisms of T cell-dependent disease pathology, treatment response, and TCR polyreactivity suggests a model in which sustained T _H 17 signaling in CD and baseline T _H 17 signaling in UC are associated with disease pathogenesis, forming a basis for a generalized understanding of IBD pathogenesis and underscoring the need for endotype-specific approaches to IBD therapy.

Rapid CD137 upregulation on alloreactive T cells upon allogeneic stimulation suggests that their selective elimination could prevent acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HCT). Here, we developed a novel aGVHD prophylactic regimen consisting of a single dose of an anti-CD137 antibody–drug conjugate (CD137-ADC) administered on the day of transplant without additional immunosuppression. The CD137-ADC depleted both human and nonhuman primate (NHP) activated T cells and proved highly effective in preventing xenogeneic aGVHD in mice receiving human peripheral blood mononuclear cells, as well as in NHP undergoing major histocompatibility complex (MHC)-haploidentical HCT. Flow cytometry analysis of NHP T cells indicated specific depletion of activated PD-1+ CD4 and CD8 T cells, while sparing naïve and PD-1−OX40+ memory T-cell subsets during the first week after HCT. CD137-ADC–treated NHP recipients demonstrated robust hematopoietic and immune reconstitution. Hallmarks of T-cell recovery after CD137-ADC, which were associated with long-term aGVHD-free survival, included reconstitution of CD4 memory T cells expressing TRAIL, terminally differentiated CD8 T cells expressing CX3CR1, and CD4 FoxP3+ regulatory T cells, cell types not expected to be involved in aGVHD pathogenesis. CD137-ADC–treated recipients demonstrated a higher risk of reactivation of rhesus lymphocryptovirus (the rhesus macaque Epstein-Barr virus analog), which was associated with reconstitution of follicular helper T cells, interferon signaling–associated memory, and γδT-cell subsets. This reactivation was controllable with rituximab administration. These results document effective depletion of alloreactive T cells and prevention of aGVHD after a single dose of CD137-ADC, suggesting that clinical translation should be carefully explored.

Regulatory T cells (T _regs ) make major contributions to immune homeostasis. Because T _reg dysfunction can lead to both allo- and autoimmunity, there is interest in correcting these disorders through T _reg adoptive transfer. Two of the central challenges in clinically deploying T _reg cellular therapies are ensuring phenotypic stability and maximizing potency. Here, we describe an approach to address both issues through the creation of OX40 ligand (OX40L)–specific chimeric antigen receptor (CAR)–T _regs under the control of a synthetic forkhead box P3 ( FOXP3 ) promoter. The creation of these CAR-T _regs enabled selective T _reg stimulation by engagement of OX40L, a key activation antigen in alloimmunity, including both graft-versus-host disease and solid organ transplant rejection, and autoimmunity, including rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus. We demonstrated that OX40L–CAR-T _regs were robustly activated in the presence of OX40L-expressing cells, leading to up-regulation of T _reg suppressive proteins without induction of proinflammatory cytokine production. Compared with control T _regs , OX40L–CAR-T _regs more potently suppressed alloreactive T cell proliferation in vitro and were directly inhibitory toward activated monocyte-derived dendritic cells (DCs). We identified trogocytosis as one of the central mechanisms by which these CAR-T _regs effectively decrease extracellular display of OX40L, resulting in decreased DC stimulatory capacity. OX40L–CAR-T _regs demonstrated an enhanced ability to control xenogeneic graft-versus-host disease compared with control T _regs without abolishing the graft-versus-leukemia effect. These results suggest that OX40L–CAR-T _regs may have wide applicability as a potent cellular therapy to control both allo- and autoimmune diseases.

We developed entangled link-augmented stretchable tissue-hydrogel (ELAST), a technology that transforms tissues into elastic hydrogels to enhance macromolecular accessibility and mechanical stability simultaneously. ELASTicized tissues are highly stretchable and compressible, which enables reversible shape transformation and faster delivery of probes into intact tissue specimens via mechanical thinning. This universal platform may facilitate rapid and scalable molecular phenotyping of large-scale biological systems, such as human organs.

In this photo essay, we present a sampling of technologies from laboratories at the forefront of whole-brain clearing and imaging for high-resolution analysis of cell populations and neuronal circuits. The data presented here were provided for the eponymous Mini-Symposium presented at the Society for Neuroscience's 2018 annual meeting.

We report on the first measurements of definitive depolarization to access gold nanorod (GNR) perturbation and visualize GNR diffusion, distribution and concentration ex vivo, in vitro and in vivo in biologically and medically relevant scenarios.

Owing to their electromagnetic properties, tunability and biocompatibility, gold nanorods are being investigated as multifunctional probes for a range of biomedical applications. However, detection beyond the reach of traditional fluorescence and two-photon approaches and quantitation of their concentration in biological tissue remain challenging tasks in microscopy. Here, we show how the size and aspect ratio that impart gold nanorods with their plasmonic properties also make them a source of entropy. We report on how depolarization can be exploited as a strategy to visualize gold nanorod diffusion and distribution in biologically relevant scenarios ex vivo, in vitro and in vivo. We identify a deterministic relation between depolarization and nanoparticle concentration. As a result, some of the most stringent experimental conditions can be relaxed, and susceptibility to artefacts is reduced, enabling microscopic and macroscopic applications.