Naive Phenotype Research Articles

Abstract 4236: A subtype-specific T-cell transcriptomic atlas reveals determinants of T-cell dysfunction in CAR T-cell therapy resistance

Abstract Chimeric Antigen Receptor (CAR) T-cell therapy has been a major success for B-cell Acute Lymphoblastic Leukemia (B-ALL), yet recent clinical trials suggest that approximately 20% of patients do not respond to therapy and 40% of initial responders relapse within a year. We hypothesize that intrinsic T-cell factors are a major contributor to early therapeutic failure to CAR T-cell therapy. In particular, since most protocols do not select for T-cell subtypes, the heterogeneity in T-cell composition likely plays a major role in CAR T-cell engineering and confounds functional and clinical correlative studies. In order to characterize the molecular phenotypes of T-cells underlying therapeutic response, we performed subtype-specific transcriptomic profiling of clinical T-cell populations in 71 patients treated with CAR T-cell therapy at the Children's Hospital of Philadelphia. We obtained clinical T-cells via clinical leukapheresis prior to CAR T-cell engineering, used FACS to separate T-cells into five subtypes per patient (Naive, Stem Cell Memory, Central Memory, Effector Memory, and Effector), and performed RNA-Sequencing on all 355 samples. To our knowledge, this is the largest and most comprehensive transcriptomic profiling of clinical T-cells in CAR T-cell therapy to date. We found that a higher proportion of CCR7+CD62L+ naive and early memory T-cell phenotypes associates with longer clinical T-cell persistence (HR: 3.25 [1.37-7.89], P = 0.006), and that combining proportions of these populations with CCR7-CD62L-CD45RO+CD95+ effector memory T-cells has the greatest prognostic value (HR: 4.82 [1.89-12.32], P < 0.001). Apoptotic signaling pathways were enriched in effector and effector memory T-cell lineages (FDR < 0.001), suggesting that programmed cell death limits the contribution of these phenotypes to long-term CAR T-cell persistence. Maintenance of naive and early memory T-cell phenotypes associates positively with expression of transcription factors TCF7 and LEF1 (FDR < 0.001) and negatively with TBX21, EOMES, and ZEB2 (FDR < 0.001). Within T-cell subtypes, mixed-effects regression and integrative network analysis identified genes and network modules that discriminate between clinical responders and non-responders. We found that higher expression of CRTAM and SLAMF6 in naive and stem cell memory T-cells associates positively with clinical CAR T-cell persistence (FDR = 0.0088-0.019), suggesting that these lineage-determining factors direct T-cell fates toward effective CAR T-cell lineages. Finally, we identify an enrichment of type I interferon response pathways among patients with poor CAR T-cell persistence (F-test P < 0.001), suggesting a role of the chronic interferon response network in T-cell dysfunction. Together, these data shed light on the critical role of pre-infusion T-cell populations in CAR T-cell therapy, and may inform clinical prognosis and the development of improved CAR T-cell therapies. Citation Format: Gregory M. Chen, Changya Chen, Rajat K. Das, Yang-Yang Ding, Bing He, Hannah Kim, David M. Barrett, Kai Tan. A subtype-specific T-cell transcriptomic atlas reveals determinants of T-cell dysfunction in CAR T-cell therapy resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4236.

Development of a closed system process for purifying naive CD8+ cells, culturing and transducing with a CD19/22 chimeric antigen receptor (CAR) to produce a clinical T memory stem cell product directed against B cell malignancies

Background & Aim Currently in the Center for Cellular Engineering at the National Institutes of Health, a clinical trial which involves purifying CD8+CD62L+CD45RA+ naive T cells, culturing and transducing with a retroviral CD19 CAR, is underway. Mononuclear cells are enriched from apheresis product by automated density gradient, followed by 3 separate Strep-tactin® magnetic microbead manual selections. The selected Naive T cells are then transduced and cultured for 7 days in gas permeable cell culture bags. The entire process is open, especially purification procedures which are extremely labor intensive, takes between 16-18 hours and is performed over two days. Therefore, a less labor intensive, more streamlined and closed system process is needed. Methods, Results & Conclusion We developed a more efficient and GMP compliant process for generating CD19 and CD22 CAR Tscm product. A large scale CD8+ selection was performed on the CliniMacs Prodigy® platform followed by sterile, closed system cell sorting on the MACSQuant Tyto® for the purification of naive T cells (targets). These purified cells were placed back on the the Prodigy for culture and transduction using an automated customized program. While these enrichments worked well for donors with relatively normal target percentages, several older donors with low targets did not reach the desired purity. We then adopted a CD8+ releasable microbead selection, performed on the CliniMacs Plus device, followed by a CD62L+ microbead selection (Miltenyi Biotec) on the CliniMacs Prodigy to further enrich for target cells. We have performed many small scale selections and sorts and can consistently achieve 80-90% purities utilizing two selections. New MACSQuant Tyto® HS cartridges are now available which could potentially cut the sorting time in half, and along with new recently developed software program to decrease user setting manipulations, resulting in decreased sorting times and increased purities. In addition, after optimizing culture conditions in the CliniMacs Prodigy, as few as 25 million naive T cells can be initiated for culture, and a naive phenotype can be retained after 7 days in culture with a single lentiviral transduction. In summary, we developed a robust GMP compliant process for Tscm purification, culture and transduction which can be used to generate CAR Tscm products.

Drug efflux transporters and metabolic enzymes in human circulating and testicular T-cell subsets: relevance to HIV pharmacotherapy.

ATP-binding cassette (ABC) drug efflux transporters and drug metabolic enzymes could reduce antiretroviral concentrations in HIV target cells. The testis has been demonstrated to be a sanctuary site, displaying suboptimal antiretroviral concentrations and persistent HIV infection. Therefore, we compared the expression and function of ABC transporters and metabolic enzymes in CD4 and CD8 T cells isolated from human testis and peripheral blood mononuclear cells (PBMCs), and assessed their expression in circulating naive and memory CD4 T-cell phenotypes. Testicular tissue and blood were collected from 15 uninfected donors undergoing gender affirmation surgery. Testicular interstitial cells were isolated by enzymatic digestion, whereas PBMCs were isolated from blood by density gradient centrifugation. The expression and/or function of ABC transporters and metabolic enzymes were examined in blood and testicular T-cell subsets by flow cytometry. ABC transporters (P-gp, BCRP, MRP1) and metabolic enzymes (CYP3A4, UGT1A1) were expressed in testicular and circulating CD4 and CD8 T cells, as well as in circulating naive, central, transitional, and effector memory T-cell phenotypes. MRP1 demonstrated lower frequencies in T cells from testis compared with PBMCs, as well as in circulating naive T cells compared with the memory T-cell phenotypes. Functional activity of P-gp and BCRP was detected in T-cell subsets from testis and PBMCs. Our findings demonstrate for the first time that antiretroviral drug efflux transporters and metabolic enzymes are functionally expressed in T-cell subsets infiltrating the human testis. These transporters and enzymes can reduce antiretroviral intracellular concentrations, potentially contributing to residual HIV replication in the testis, and negatively impact HIV cure strategies.

Abstract PR3: Development of a transcriptomic T-cell atlas highlights the differential role of T-cell subpopulations in CAR T-cell therapy resistance

Abstract Chimeric antigen receptor (CAR) T-cell therapy has been a major breakthrough in B-cell acute lymphoblastic leukemia (B-ALL), yet therapeutic resistance remains an important clinical challenge. Given that approximately 20% of patients do not respond to therapy and 40% of initial responders relapse within a year, there is a pressing need to understand the basis of therapy resistance in order to identify prognostic biomarkers and improve the therapeutic modality. We hypothesize that intrinsic T-cell factors are a major contributor to early therapeutic failure to CAR T-cell therapy. Most protocols do not select for specific T-cell subtypes, leading to the use of heterogeneous T-cell populations for CAR T-cell engineering. In order to characterize the molecular phenotypes of T cells underlying therapeutic response, we performed subtype-specific transcriptomic profiling of clinical T-cell populations in patients subsequently treated with anti-CD19 CAR T-cell therapy. We identified 71 patients with B-ALL treated with CAR T-cell therapy at the Children’s Hospital of Philadelphia, and obtained T cells via clinical leukapheresis prior to CAR T-cell engineering. We used FACS to separate T cells into five subtypes per patient (Naive, Stem Cell Memory, Central Memory, Effector Memory, and Effector), and performed bulk RNA-sequencing on all 355 samples. To our knowledge, this is the largest and most comprehensive transcriptomic profiling of clinical T cells in CAR T-cell therapy to date. We find that a higher proportion of CCR7+CD62L+ naive and early memory T-cell phenotypes associates with longer clinical T-cell persistence (HR=3.25 [1.37-7.89], p=0.006), and that combining proportions of these populations with CCR7−CD62L−CD45RO+CD95+ effector memory T cells has the greatest prognostic value (HR: 4.82 [1.89-12.32]; p<0.001). We identified global transcriptomic markers of early and differentiated T-cell populations and identified differentially expressed genes that discriminated between clinical responders and nonresponders. Single-sample gene set enrichment analysis identified an enrichment of T-cell exhaustion pathways among the clinical nonresponders, which was predominant in the early lineage T-cell phenotypes (p=0.018-0.095) but not in the effector T-cell phenotypes (p=0.15-0.37). Together, these data shed light on the critical role of preinfusion T-cell populations in CAR T-cell therapy, and may inform clinical prognosis and the development of improved CAR T-cell therapies. This abstract is also being presented as Poster A65. Citation Format: Gregory M. Chen, Changya Chen, Rajat K. Das, Yang-Yang Ding, Bing He, Hannah Kim, David M. Barrett, Kai Tan. Development of a transcriptomic T-cell atlas highlights the differential role of T-cell subpopulations in CAR T-cell therapy resistance [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr PR3.

Enhancing the Efficacy of Stem Cell Therapy with Glycosaminoglycans.

SummaryHuman mesenchymal stem cell (hMSC) therapy offers significant potential for osteochondral regeneration. Such applications require their ex vivo expansion in media frequently supplemented with fibroblast growth factor 2 (FGF2). Particular heparan sulfate (HS) fractions stabilize FGF2-FGF receptor complexes. We show that an FGF2-binding HS variant (HS8) accelerates the expansion of freshly isolated bone marrow hMSCs without compromising their naivety. Importantly, the repair of osteochondral defects in both rats and pigs is improved after treatment with HS8-supplemented hMSCs (MSCHS8), when assessed histologically, biomechanically, or by MRI. Thus, supplementing hMSC culture media with an HS variant that targets endogenously produced FGF2 allows the elimination of exogenous growth factors that may adversely affect their therapeutic potency.

Understanding the Heterogeneous Population of Age-Associated B Cells and Their Contributions to Autoimmunity and Immune Response to Pathogens.

In humans and mice, susceptibility to infections and autoimmunity increases with age due to age-associated changes in innate and adaptive immune responses. Aged innate cells are also less active, leading to decreased naive T- and B-cell responses. Aging innate cells contribute to an overall heightened inflammatory environment. Naive T and B cells undergo cell-intrinsic age-related changes that result in reduced effector and memory responses. However, previously established B- and T-cell memory responses persist with age. One dramatic change is the appearance of a newly recognized population of age-associated B cells (ABCs) that has a unique cluster of differentiation (CD)21-CD23- phenotype. Here, we discuss the discovery and origins of the naive phenotype immunoglobulin (Ig)D+ versus activated CD11c+T-bet+ ABCs, with a focus on protective and pathogenic properties. In humans and mice, antigen-experienced CD11c+T-bet+ ABCs increase with autoimmunity and appear in response to bacterial and viral infections. However, our analyses indicate that CD21-CD23- ABCs include a resting, naive, progenitor ABC population that expresses IgD. Similar to generation of CD11c+T-bet+ ABCs, naive ABC response to pathogens depends on toll-like receptor stimulation, making this a key feature of ABC activation. Here, we put forward a potential developmental map of distinct subsets from putative naive ABCs. We suggest that defining signals that can harness the naive ABC response may contribute to protection against pathogens in the elderly. CD11c+T-bet+ ABCs may be useful targets for therapeutic strategies to counter autoimmunity.

Dysregulated T helper type 1 (Th1) and Th17 responses in elderly hospitalised patients with infection and sepsis.

ObjectiveThe role of Th1 and Th17 lymphocyte responses in human infection and sepsis of elderly patients has yet to be clarified.DesignA prospective observational study of patients with sepsis, infection only and healthy controls.SettingThe acute medical wards and intensive care units in a 1000 bed university hospital.Patients32 patients with sepsis, 20 patients with infection, and 20 healthy controls. Patients and controls were older than 65 years of age. Patients with recognised underlying immune compromise were excluded.MethodsPhenotype, differentiation status and cytokine production by T lymphocytes were determined by flow cytometry.MeasurementsThe differentiation states of circulating CD3+, CD4+, and CD8+ T cells were characterised as naive (CD45RA+, CD197+), central memory (CD45RA-, CD197+), effector memory (CD45RA-, CD197-), or terminally differentated (CD45RA+, CD197-). Expression of IL-12 and IL-23 receptors, and the transcription factors T-bet and RORγt, was analysed in circulating T lymphocytes. Expression of interferon- γ and IL-17A were analysed following stimulation in vitro.ResultsCD4+ T cells from patients with infection predominantly expressed effector-memory or terminally differentiated phenotypes but CD4+ T cells from patients with severe sepsis predominantly expressed naive phenotypes (p<0.0001). CD4+ T cells expressing IL-23 receptor were lower in patients with sepsis compared to patients with infection alone (p = 0.007). RORγt expression by CD4+ T cells was less frequent in patients with sepsis (p<0.001), whereas T-bet expressing CD8+ T cells that do not express RORγt was lower in the sepsis patients.HLA-DR expression by monocytes was lower in patients with sepsis. In septic patients fewer monocytes expressed IL-23.ConclusionPersistent failure of T cell activation was observed in patients with sepsis. Sepsis was associated with attenuated CD8+Th1 and CD4+Th17 based lymphocyte response.

Recovery of central memory and naive peripheral T cells in Follicular Lymphoma patients receiving rituximab-chemotherapy based regimen

Preclinical models and clinical studies have shown that anti-CD20-based treatment has multifaceted consequences on T-cell immunity. We have performed a prospective study of peripheral T-cell compartment in FL patients, all exhibiting high tumor burden and receiving rituximab-chemotherapy-based regimen (R-CHOP). Before treatment, FL patients harbor low amounts of peripheral naive T cells, but high levels of CD4+ TEM, CD4+ Treg and CD8+ TEMRA subsets and significant amounts of CD38+ HLA-DR+ activated T cells. A portion of these activated/differentiated T cells also expressed PD-1 and/or TIGIT immune checkpoints. Hierarchical clustering of phenotyping data revealed that 5/8 patients with only a partial response to R-CHOP induction therapy or with disease progression segregate into a group exhibiting a highly activated/differentiated T cell profile and a markedly low proportion of naive T cells before treatment. Rituximab-based therapy induced a shift of CD4+ and CD8+ T cells toward a central memory phenotype and of CD8+ T cells to a naive phenotype. In parallel, a decrease in the number of peripheral T cells expressing both PD-1 and TIGIT was detected. These observations suggest that the standard rituximab-based therapy partially reverts the profound alterations observed in T-cell subsets in FL patients, and that blood T-cell phenotyping could provide a better understanding of the mechanisms of rituximab-based treatment.

A Subset of Mycobacteria-Specific CD4+ IFN-γ+ T Cell Expressing Naive Phenotype Confers Protection against Tuberculosis Infection in the Lung.

Failure of the most recent tuberculosis (TB) vaccine trial to boost bacillus Calmette-Guérin-mediated anti-TB immunity despite the induction of Th1-specific central memory cell and effector memory cell responses highlights the importance of identifying optimal T cell targets for protective vaccines. In this study, we describe a novel, Mycobacterium tuberculosis-specific IFN-γ+CD4+ T cell population expressing surface markers characteristic of naive-like memory T cells (TNLM), which were induced in both human (CD45RA+CCR7+CD27+CD95-) and murine (CD62L+CD44-Sca-1+CD122-) systems in response to mycobacteria. In bacillus Calmette-Guérin-vaccinated subjects and those with latent TB infection, TNLM were marked by the production of IFN-γ but not TNF-α and identified by the absence of CD95 expression and increased surface expression CCR7, CD27, the activation markers T-bet, CD69, and the survival marker CD74. Increased tetramer-positive TNLM frequencies were noted in the lung and spleen of ESAT-61-20-specific TCR transgenic mice at 2 wk postinfection with M. tuberculosis and progressively decreased at later time points, a pattern not seen with TNF-α+CD4+ T cells expressing naive cell surface markers. Importantly, adoptive transfer of highly purified TNLM alone, from vaccinated ESAT-61-20-specific TCR transgenic mice, conferred equivalent protection against M. tuberculosis infection in the lungs of Rag-/- mice when compared with total memory populations (central and effector memory cells). Thus, TNLM may represent a memory T cell population that, if optimally targeted, may significantly improve future TB vaccine responses.

CMIP is a negative regulator of T cell signaling.

Upon their interaction with cognate antigen, T cells integrate different extracellular and intracellular signals involving basal and induced protein–protein interactions, as well as the binding of proteins to lipids, which can lead to either cell activation or inhibition. Here, we show that the selective T cell expression of CMIP, a new adapter protein, by targeted transgenesis drives T cells toward a naïve phenotype. We found that CMIP inhibits activation of the Src kinases Fyn and Lck after CD3/CD28 costimulation and the subsequent localization of Fyn and Lck to LRs. Video microscopy analysis showed that CMIP blocks the recruitment of LAT and the lipid raft marker cholera toxin B at the site of TCR engagement. Proteomic analysis identified several protein clusters differentially modulated by CMIP and, notably, Cofilin-1, which is inactivated in CMIP-expressing T cells. Moreover, transgenic T cells exhibited the downregulation of GM3 synthase, a key enzyme involved in the biosynthesis of gangliosides. These results suggest that CMIP negatively impacts proximal signaling and cytoskeletal rearrangement and defines a new mechanism for the negative regulation of T cells that could be a therapeutic target.

Dimorphism in the T-cell receptor constant region affects T-cell function, phenotype and HIV outcome.

CD8 T cells recognize human leukocyte antigen-peptide complex through the T-cell receptor. Although amino acid variation in T-cell receptor variable chains often affects antigen specificity, dimorphism in the beta chain constant region (TRBC1 and TRBC2) is not thought to affect T-cell function. A recent study suggested that adoptive transfer of TRBC1-specific chimeric antigen-receptor-T cells provided an option for T-cell leukemia therapy that preserved T-cell immunity in the TRBC2 subset. This raises an important question as to whether TRBC1T cells are qualitatively different from TRBC2T cells. Cross-sectional study. Sixty-six antiretroviral therapy-naive HIV-infected individuals, including 19 viraemic controllers and 47 noncontrollers, were enrolled. Peripheral blood mononuclear cells were isolated for T-cell functional assays, tetramer analyses, TRBC1 staining and immunophenotyping. Viraemic controllers had a higher proportion of circulating TRBC1T cells than noncontrollers, raising the possibility that TRBC1T cells might be associated with HIV control. TRBC1T cells also showed more functional T-cell responses against both HIV and cytomegalovirus (P < 0.01). The immunophenotypes of TRBC1-bearing T cells were skewed towards naive and central memory phenotypes, whereas the majority of TRBC2-expressing T cells were terminally differentiated. Inverse correlations were observed between %TRBC1T cells and HIV plasma viral load, which was most pronounced for CD8 T cells (r = -0.7096, P = 0.00002357). These data suggest that TRBC1T-cell responses are of better quality than their TRBC2 counterparts, which should be considered in immunotherapeutic strategies for HIV infection. Conversely, depletion of TRBC1T cells as part of the treatment of TRBC1 T-cell malignancies may lead to compromised T-cell response quality.

Reference intervals for lymphocyte subsets in preterm and term neonates without immune defects

In 6.5years of newborn screening for severe combined immunodeficiency in California, 3,252,156 infants had DNA from dried blood spots (DBSs) assayed for T-cell receptor excision circles. Infants with T-cell receptor excision circle values of less than a designated cutoff on a single DBS, 2 DBS samples with insufficient PCR amplification, or known genetic risk of immunodeficiency had peripheral blood complete blood counts and lymphocyte subsets assayed in a single flow cytometry laboratory. Cases in which immune defects were ruled out were available for analysis. We sought to determine reference intervals for lymphocyte subsets in racially/ethnically diverse preterm and term newborns who proved to be unaffected by any T-lymphopenic immune disorder. Effective gestational age (GA) was defined as GA at birth plus postnatal age at the time of sample collection. After determining exclusion criteria, we analyzed demographic and clinical information, complete and differential white blood cell counts, and lymphocyte subsets for 301 infants, with serial measurements for 33 infants. Lymphocyte subset measurements included total T cells, helper and cytotoxic T-cell subsets, naive and memory phenotype of each T-cell subset, B cells, and natural killer cells. Reference intervals were generated for absolute numbers and lymphocyte subsets from infants with effective GAs of 22 to 52weeks. Sex and ethnicity were not significant determinants of lymphocyte subset counts in this population. Lymphocyte counts increased postnatally. This study provides a baseline for interpreting comprehensive lymphocyte data in preterm and term infants, aiding clinicians to determine which newborns require further evaluations for immunodeficiency.

Exploring novel targets for CAR-T therapy for prostate cancer

Background & Aim Prostate Cancer (PCa) affects one in six men. Treatment options for men with localized disease include radical prostatectomy or brachytherapy. Unfortunately, patients may also present with advanced PCa which invariably progresses to castration-resistant prostate cancer (CRPC), which is lethal due to treatment resistance. There is an unmet need in CRPC to develop new therapies that improve outcome. Harnessing the immune system offers great potential in this regard. In this study, we have identified novel antigens as targets for immunotherapy in men with prostate cancer. These include the carbohydrate antigen Lewis Y, and the stress response chaperone protein GRP-78. Lewis Y antigen is highly expressed in prostate cancer, particularly in higher grade primary tumours and in metastatic and hormone refractory disease. GRP78 is also expressed in prostate cancer in men with more aggressive disease. Methods, Results & Conclusion We generated a second-generation CAR targeting either Lewis Y or GRP-78 with a truncated CD34-tag for purification. Because the differentiation and phenotype of the CAR-T product may have a critical impact on the post-infusion antitumor effect and persistence of the cells, we also investigated the phenotype of CAR-T product with different γc cytokines, i.e. IL-2, IL-7 and IL-15, to generate early differentiation stage cells with less exhaustion phenotypes. In the optimized protocol, by using the retroviral transduction system, starting from 3 × 107 PBMC, we're now able to produce approximately 109 bulk CAR-T cells or 5 × 108 purified CAR-T cells with over 98% purity, within 7-10 days. Furthermore, most of these cells remained early naive or memory-like phenotype. These engineered T cells are capable to eradicate prostate cancer tumour cells in vitro and to secrete TNF-α and IFN-γ cytokines. The in vivo study also showed that the infused cells can reduce the tumour burden and persist post infusion. Our results demonstrate that the novel CAR-T cells exhibited specific and efficient tumoricidal effect against prostate cancer cells. Furthermore, by introducing the CD34-tag, with the optimized protocol, we're now able to produce CAR-T cells with high purity and more early differentiation stage in a relatively shorter time. This may further benefit the clinical application of CAR-T therapy, by reducing the total number of the infused cells and shortening the waiting period for CAR-T production.

The Cell-Surface Marker Sushi Containing Domain 2 Facilitates Establishment of Human Naive Pluripotent Stem Cells.

SummaryRecently naive human pluripotent stem cells (hPSCs) have been described that relate to an earlier stage of development than conventional hPSCs. Naive hPSCs remain challenging to generate and authenticate, however. Here we report that Sushi Containing Domain 2 (SUSD2) is a robust cell-surface marker of naive hPSCs in the embryo and in vitro. SUSD2 transcripts are enriched in the pre-implantation epiblast of human blastocysts and immunostaining shows localization of SUSD2 to KLF17-positive epiblast cells. SUSD2 mRNA is strongly expressed in naive hPSCs but is negligible in other hPSCs. SUSD2 immunostaining of live or fixed cells provides unambiguous discrimination of naive versus conventional hPSCs. SUSD2 staining or flow cytometry enable monitoring of naive hPSCs in maintenance culture, and their isolation and quantification during resetting of conventional hPSCs or somatic cell reprogramming. Thus SUSD2 is a powerful non-invasive tool for reliable identification and purification of the naive hPSC phenotype.

Mechanisms of Abrupt Loss of Virus Control in a Cohort of Previous HIV Controllers.

Elite and viremic HIV controllers are able to control their HIV infection and maintain undetectable or low-level viremia in the absence of antiretroviral treatment. Despite extensive studies, the immune factors responsible for such exclusive control remain poorly defined. We identified a cohort of 14 HIV controllers that suffered an abrupt loss of HIV control (LoC) to investigate possible mechanisms and virological and immunological events related to the sudden loss of control. The in-depth analysis of these subjects involved the study of cell tropism of circulating virus, evidence for HIV superinfection, cellular immune responses to HIV, as well as an examination of viral adaptation to host immunity by Gag sequencing. Our data demonstrate that a poor capacity of T cells to mediate in vitro viral suppression, even in the context of protective HLA alleles, predicts a loss of viral control. In addition, the data suggest that inefficient viral control may be explained by an increase of CD8 T-cell activation and exhaustion before LoC. Furthermore, we detected a switch from C5- to X4-tropic viruses in 4 individuals after loss of control, suggesting that tropism shift might also contribute to disease progression in HIV controllers. The significantly reduced inhibition of in vitro viral replication and increased expression of activation and exhaustion markers preceding the abrupt loss of viral control may help identify untreated HIV controllers that are at risk of losing control and may offer a useful tool for monitoring individuals during treatment interruption phases in therapeutic vaccine trials.IMPORTANCE A few individuals can control HIV infection without the need for antiretroviral treatment and are referred to as HIV controllers. We have studied HIV controllers who suddenly lose this ability and present with high in vivo viral replication and decays in their CD4+ T-cell counts to identify potential immune and virological factors that were responsible for initial virus control. We identify in vitro-determined reductions in the ability of CD8 T cells to suppress viral control and the presence of PD-1-expressing CD8+ T cells with a naive immune phenotype as potential predictors of in vivo loss of virus control. The findings could be important for the clinical management of HIV controller individuals, and it may offer an important tool to anticipate viral rebound in individuals in clinical studies that include combination antiretroviral therapy (cART) treatment interruptions and which, if not treated quickly, could pose a significant risk to the trial participants.

Human Bone Marrow Derived Mesenchymal Stromal Cells Enhance the Number and Function of Umbilical Cord Blood Peripheral Tregs during IL-2 Driven Ex Vivo Expansion

Tumor growth factor β (TGF-β)-induced peripheral regulatory T cells (pTreg) are a promising therapeutic cell source that exhibit Foxp3 expression and suppressive functions similar to natural regulatory T cells. Nonetheless, their clinical potential is limited by the instability of Foxp3 expression and T cell exhaustion that occurs during ex vivo expansion. We postulated that mesenchymal stromal cells (MSCs) could enhance the number, function and Foxp3 expression stability of pTregs during IL-2 driven 21 day expansion due to their diverse immunomodulatory properties. In this study, we observed that use of a human bone marrow mesenchymal stromal cells (hBM-MSC) platform significantly enhanced the number of pTreg during IL-2 driven 21 day ex vivo expansion vs. standard suspension culture condition (MSC platform: 80.2 x 106 vs. IL2/media: 39.3 x 106, n=6; p<0.01). Also the number of pTreg expressing a naive phenotype (CD4+CD45RA+ and CD4+CD62L+ ) were significantly increased (CD45RA+; MSC platform: 74.4 ± 1.6 x 106 vs. IL2/media: 45.9 ± 2.9 x 106, n=6, p<0.001; CD62L+; MSC platform: 79.1 ± 1.3 x 106 vs. IL2/media: 54.5 ± 2.1 x 106, n=6, p<0.001), as well as stability of Foxp3 expression (IL-2/media: 88.2 ± 1.7% vs. MSC platform: 96.2 ± 1.1%, n=7; p<0.05). In addition, pTreg suppressive function was noted to be more potent during 21 day IL-2 driven ex vivo expansion compared to standard IL-2/media culture condition (MSC platform: 79% vs. media: 35% inhibition of T cell proliferation in 10:1 ratio, n=6; p<0.01). pTreg expanded over a hBM-MSC platform exhibited higher surface CD25, CTLA-4, and ICOS MFI expression (CD25; MSC platform: 1410 vs. Media: 774; p<0.001, CTLA-4; MSC platform: 1084 vs. Media: 318; p<0.001, ICOS; MSC platform: 4386 vs. Media: 2641, p<0.01, n=6). Notably, hBM-MSC enhancement of pTreg ex vivo expansion requires direct cell-cell contact, as Foxp3 expression in pTreg was not enhanced by hBM-MSC conditioned media (CM:73.4 ± 6.8% vs. MSC platform: 96.2 ± 1.0%, p<0.001; and IL2/media: 88.8 ± 1.6% vs. MSC platform: 96.2 ± 1.0%, p<0.01) nor in a trans-well culture experiments (Transwell: 83.4 ± 2.5% vs. IL2/media: 88.8 ± 1.6%; and Transwell: 83.4 ± 2.5% vs. MSC platform: 96.2 ± 1.0%, p<0.01). Importantly, optical sectioning microscopy and flow cytometry revealed that hBM-MSC supports Treg number and function via direct contact-dependent mitochondrial transfer (Figure 1A-B). Cytochalasin B treatment blocked mitochondrial transfer, suggesting that tunneling nanotubes (TNT) facilitate mitochondrial transfer from hBM-MSC to pTreg during IL-2 driven ex vivo expansion (Mock: 2208 ± 122.1 vs. Cyto B: 923.8 ± 89 MFI, n=6, p<0.0001). Moreover, the quantity of ATP (n=6; p<0.01) mitochondrial potential of pTreg (MSC platform: 9010 ± 224.5 vs. media: 7316 ± 122.7 MFI, n=6; p<0.01) were significantly enhanced in pTreg during IL-2 driven ex vivo expansion over a hBM-MSC platform. Taken together, hBM-MSC significantly improves the number, maturation, and function of pTreg during 21 day IL-2 driven ex vivo expansion. We have identified one key mechanism of action of hBM-MSC underlying these favorable effects on pTreg during ex vivo expansion to be mitochondrial transfer via TNT. Notably, these studies identify a novel role of hBM-MSC to overcome current limitations in IL-2/media suspension culture conditions including T cell senescence, and loss of Foxp3 expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Chemically reversible hydrogels

Dynamic Materials![Figure][1] Illustration of triggered superstructure changes CREDIT: MARK SENIW, SIMPSON QUERREY INSTITUTE, NORTHWESTERN UNIVERSITY The dynamic reorganization of some cellular biopolymers in response to signals has inspired efforts to create artificial materials with similar properties. Freeman et al. created hydrogels based on peptide amphiphiles that can bear DNA strands that assemble into superstructures and that disassemble in response to chemical triggers. The addition of DNA conjugates induced transitions from micelles to fibers and bundles of fibers. The resulting hydrogels were used as an extracellular matrix mimic for cultured cells. Switching the hydrogel between states also switched astrocytes between their reactive and naive phenotypes. Science , this issue p. [808][2] [1]: pending:yes [2]: /lookup/doi/10.1126/science.aat6141

Maintained thymic output of conventional and regulatory T cells during human pregnancy

Maintained thymic output of conventional and regulatory T cells during human pregnancy

Functional characterization of reappearing B cells after anti-CD20 treatment of CNS autoimmune disease

The anti-CD20 antibody ocrelizumab, approved for treatment of multiple sclerosis, leads to rapid elimination of B cells from the blood. The extent of B cell depletion and kinetics of their recovery in different immune compartments is largely unknown. Here, we studied how anti-CD20 treatment influences B cells in bone marrow, blood, lymph nodes, and spleen in models of experimental autoimmune encephalomyelitis (EAE). Anti-CD20 reduced mature B cells in all compartments examined, although a subpopulation of antigen-experienced B cells persisted in splenic follicles. Upon treatment cessation, CD20+ B cells simultaneously repopulated in bone marrow and spleen before their reappearance in blood. In EAE induced by native myelin oligodendrocyte glycoprotein (MOG), a model in which B cells are activated, B cell recovery was characterized by expansion of mature, differentiated cells containing a high frequency of myelin-reactive B cells with restricted B cell receptor gene diversity. Those B cells served as efficient antigen-presenting cells (APCs) for activation of myelin-specific T cells. In MOG peptide-induced EAE, a purely T cell-mediated model that does not require B cells, in contrast, reconstituting B cells exhibited a naive phenotype without efficient APC capacity. Our results demonstrate that distinct subpopulations of B cells differ in their sensitivity to anti-CD20 treatment and suggest that differentiated B cells persisting in secondary lymphoid organs contribute to the recovering B cell pool.

Epigenetic treatment of multiple myeloma mediates tumor intrinsic and extrinsic immunomodulatory effects

ABSTRACTImmune evasion is an important driver of disease progression in the plasma cell malignancy multiple myeloma. Recent work highlights the potential of epigenetic modulating agents as tool to enhance anti-tumor immunity. The immune modulating effects of the combination of a DNA methyltransferase inhibitor and a histone deacetylase inhibitor in multiple myeloma is insufficiently characterized. Therefore, we used the murine immunocompetent 5T33MM model to investigate hallmarks of immunogenic cell death as well as alterations in the immune cell constitution in the bone marrow of diseased mice in response to the DNA methyltransferase inhibitor decitabine and the histone deacetylase inhibitor quisinostat. Vaccination of mice with 5T33 cells treated with epigenetic compounds delayed tumor development upon a subsequent tumor challenge. In vitro, epigenetic treatment induced ecto-calreticulin and CD47, as well as a type I interferon response. Moreover, treated 5T33vt cells triggered dendritic cell maturation. The combination of decitabine and quisinostat in vivo resulted in combinatory anti-myeloma effects. In vivo, epigenetic treatment increased tumoral ecto-calreticulin and decreased CD47 and PD-L1 expression, increased dendritic cell maturation and reduced CD11b positive cells. Moreover, epigenetic treatment induced a temporal increase in presence of CD8-positive and CD4-positive T cells with naive and memory-like phenotypes based on CD62L and CD44 expression levels, and reduced expression of exhaustion markers PD-1 and TIM3. In conclusion, a combination of a DNA methyltransferase inhibitor and a histone deacetylase inhibitor increased the immunogenicity of myeloma cells and altered the immune cell constitution in the bone marrow of myeloma-bearing mice.