Multiple Signaling Roles of CD3ε and Its Application in CAR-T Cell Therapy.

A T cell receptor (TCR) mediates antigen-induced signaling through its associated CD3ε, δ, γ, and ζ, but the contributions of different CD3 chains remain elusive. Using quantitative mass spectrometry, we simultaneously quantitated the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of all CD3 chains upon TCR stimulation. A subpopulation of CD3ε ITAMs was mono-phosphorylated, owing to Lck kinase selectivity, and specifically recruited the inhibitory Csk kinase to attenuate TCR signaling, suggesting that TCR is a self-restrained signaling machinery containing both activating and inhibitory motifs. Moreover, we found that incorporation of the CD3ε cytoplasmic domain into a second-generation chimeric antigen receptor (CAR) improved antitumor activity of CAR-T cells. Mechanistically, the Csk-recruiting ITAM of CD3ε reduced CAR-T cytokine production whereas the basic residue rich sequence (BRS) of CD3ε promoted CAR-T persistence via p85 recruitment. Collectively, CD3ε is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy to design next-generation CAR.

Chimeric Antigen Receptor Designed to Prevent Ubiquitination and Downregulation Showed Durable Antitumor Efficacy.

Clinical evidence suggests that poor persistence of chimeric antigen receptor-T cells (CAR-T) in patients limits therapeutic efficacy. Here, we designed a CAR with recyclable capability to promote in vivo persistence and to sustain antitumor activity. We showed that the engagement of tumor antigens induced rapid ubiquitination of CARs, causing CAR downmodulation followed by lysosomal degradation. Blocking CAR ubiquitination by mutating all lysines in the CAR cytoplasmic domain (CARKR) markedly repressed CAR downmodulation by inhibiting lysosomal degradation while enhancing recycling of internalized CARs back to the cell surface. Upon encountering tumor antigens, CARKR-T cells ameliorated the loss of surface CARs, which promoted their long-term killing capacity. Moreover, CARKR-T cells containing 4-1BB signaling domains displayed elevated endosomal 4-1BB signaling that enhanced oxidative phosphorylation and promoted memory T cell differentiation, leading to superior persistence in vivo. Collectively, our study provides a straightforward strategy to optimize CAR-T antitumor efficacy by redirecting CAR trafficking.

Endogenous Signaling Molecule Activating (ESMA) CARs: A Novel CAR Design Showing a Favorable Risk to Potency Ratio for the Treatment of Triple Negative Breast Cancer.

As chimeric antigen receptor (CAR) T cell therapy continues to gain attention as a valuable treatment option against different cancers, strategies to improve its potency and decrease the side effects associated with this therapy have become increasingly relevant. Herein, we report an alternative CAR design that incorporates transmembrane domains with the ability to recruit endogenous signaling molecules, eliminating the need for stimulatory signals within the CAR structure. These endogenous signaling molecule activating (ESMA) CARs triggered robust cytotoxic activity and proliferation of the T cells when directed against the triple-negative breast cancer (TNBC) cell line MDA-MB-231 while exhibiting reduced cytokine secretion and exhaustion marker expression compared to their cognate standard second generation CARs. In a NOD SCID Gamma (NSG) MDA-MB-231 xenograft mouse model, the lead candidate maintained longitudinal therapeutic efficacy and an enhanced T cell memory phenotype. Profound tumor infiltration by activated T cells repressed tumor growth, further manifesting the proliferative capacity of the ESMA CAR T cell therapy. Consequently, ESMA CAR T cells entail promising features for improved clinical outcome as a solid tumor treatment option.

CAR T-Cell Persistence Correlates with Improved Outcome in Patients with B-Cell Lymphoma.

Chimeric antigen receptor (CAR) T-cell therapy has led to profound and durable tumor responses in a relevant subset of patients with relapsed/refractory (r/r) B-cell lymphomas. Still, some patients show insufficient benefit or relapse after CAR T-cell therapy. We performed a retrospective study to investigate the correlation between CAR T-cell persistence in the peripheral blood (PB) at 6 months, assessed by droplet digital PCR (ddPCR), with CAR T-cell treatment outcome. 92 patients with r/r B-cell lymphomas were treated with CD19-targeting CAR T-cell therapies at our institution between 01/2019-08/2022. Six months post-treatment, 15 (16%) patients had no detectable circulating CAR-T constructs by ddPCR. Patients with CAR T-cell persistence had a significantly higher CAR T-cell peak (5432 vs. 620 copies/ug cfDNA, p = 0.0096), as well as higher incidence of immune effector cell-associated neurotoxicity syndrome (37% vs. 7%, p = 0.0182). After a median follow-up of 8.5 months, 31 (34%) patients relapsed. Lymphoma relapses were less frequent among patients with CAR T-cell persistence (29% vs. 60%, p = 0.0336), and CAR T-cell persistence in the PB at 6 months was associated with longer progression-free survival (PFS) (HR 2.79, 95% CI: 1.09-7.11, p = 0.0319). Moreover, we observed a trend towards improved overall survival (OS) (HR 1.99, 95% CI: 0.68-5.82, p = 0.2092) for these patients. In our cohort of 92 B-cell lymphomas, CAR T-cell persistence at 6 months was associated with lower relapse rates and longer PFS. Moreover, our data confirm that 4-1BB-CAR T-cells have a longer persistence as compared to CD-28-based CAR T-cells.

Composite CD79A/CD40 co-stimulatory endodomain enhances CD19CAR-T cell proliferation and survival.

Adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have led to impressive clinical outcomes in B cell malignancies. Beyond induction of remission, the persistence of CAR-T cells is required to prevent relapse and provide long-term disease control. To improve CAR-T cell function and persistence, we developed a composite co-stimulatory domain of a B cell signaling moiety, CD79A/CD40, to induce a nuclear translocating signal, NF-κB, to synergize with other T cell signals and improve CAR-T cell function. CD79A/CD40 incorporating CD19CAR-T cells (CD19.79a.40z) exhibited higher NF-κB and p38 activity upon CD19 antigen exposure compared with the CD28 or 4-1BB incorporating CD19CAR-T cells (CD19.28z and CD19.BBz). Notably, we found that CD19.79a.40z CAR-T cells continued to suppress CD19+ target cells throughout the co-culture assay, whereas a tendency for tumor growth was observed with CD19.28z CAR-T cells. Moreover, CD19.79a.40z CAR-T cells exhibited robust T cell proliferation after culturing with CD19+ target cells, regardless of exogenous interleukin-2. In terms of in vivo efficiency, CD19.79a.40z demonstrated superior anti-tumor activity and in vivo CAR-T cell proliferation compared with CD19.28z and CD19.BBz CD19CAR-T cells in Raji-inoculated mice. Our data demonstrate that the CD79A/CD40 co-stimulatory domain endows CAR-T cells with enhanced proliferative capacity and improved anti-tumor efficacy in a murine model.

Preferential Small Intestine Homing and Persistence of CD8 T Cells in Rhesus Macaques Achieved by Molecularly Engineered Expression of CCR9 and Reduced Ex Vivo Manipulation.

Adoptive cell transfer (ACT) is a powerful experimental approach to directly study T-cell-mediated immunity in vivo In the rhesus macaque AIDS virus model, infusing simian immunodeficiency virus (SIV)-infected animals with CD8 T cells engineered to express anti-SIV T-cell receptor specificities enables direct experimentation to better understand antiviral T-cell immunity in vivo Limiting factors in ACT experiments include suboptimal trafficking to, and poor persistence in, the secondary lymphoid tissues targeted by AIDS viruses. Previously, we redirected CD8 T cells to B-cell follicles by ectopic expression of the CXCR5 homing protein. Here, we modify peripheral blood mononuclear cell (PBMC)-derived CD8 T cells to express the CCR9 chemokine receptor, which induces preferential homing of the engineered cells to the small intestine, a site of intense early AIDS virus replication and pathology in rhesus macaques. Additionally, we increase in vivo persistence and overall systemic distribution of infused CD8 T cells, especially in secondary lymphoid tissues, by minimizing ex vivo culture/manipulation, thereby avoiding the loss of CD28+/CD95+ central memory T cells by differentiation in culture. These proof-of-principle results establish the feasibility of preferentially localizing PBMC-derived CD8 T cells to the small intestine and enables the direct experimental ACT-based assessment of the potential role of the quality and timing of effective antiviral CD8 T-cell responses to inhibit viral infection and subsequent replication in small intestine CD4 T cells. More broadly, these results support the engineered expression of homing proteins to direct CD8 T cells to target tissues as a means for both experimental and potential therapeutic advances in T-cell immunotherapies, including cancer.IMPORTANCEAdoptive cell transfer (ACT) of T cells engineered with antigen-specific effector properties can deliver targeted immune responses against malignancies and infectious diseases. Current T-cell-based therapeutic ACT relies on circulatory distribution to deliver engineered T cells to their targets, an approach which has proven effective for some leukemias but provided only limited efficacy against solid tumors. Here, engineered expression of the CCR9 homing receptor redirected CD8 T cells to the small intestine in rhesus macaque ACT experiments. Targeted homing of engineered T-cell immunotherapies holds promise to increase the effectiveness of adoptively transferred cells in both experimental and clinical settings.

Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells.

Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (TSCM) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR+ T cells with preserved TSCM potential using the Sleeping Beauty platform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19+ leukemia. Long-lived T cells were CD45ROnegCCR7+CD95+, phenotypically most similar to TSCM, and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR+ T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials.

Strategies for enhancing CAR T cell expansion and persistence in HIV infection.

Chimeric Antigen Receptor (CAR) T cell therapies are tremendously successful in hematological malignancies and show great promise as treatment and curative strategy for HIV. A major determinant for effective CAR T cell therapy is the persistence of CAR T cells. Particularly, antigen density and target cell abundance are crucial for the engagement, engraftment, and persistence of CAR T cells. The success of HIV-specific CAR T cells is challenged by limited antigen due to low cell surface expression of viral proteins and the scarcity of chronically infected cells during antiretroviral therapy. Several strategies have been explored to increase the efficacy of CAR T cells by enhancing expansion and persistence of the engineered cells. This review highlights the challenges of designing CAR T cells against HIV and other chronic viral infections. We also discuss potential strategies to enhance CAR T cell expansion and persistence in the setting of low antigen exposure.

Tumor-Specific CD8+ T Cells from the Bone Marrow Resist Exhaustion and Exhibit Increased Persistence in Tumor-Bearing Hosts as Compared to Tumor Infiltrating Lymphocytes.

Immunotherapy is now an integral aspect of cancer therapy. Strategies employing adoptive cell therapy (ACT) have seen the establishment of chimeric antigen receptor (CAR)-T cells using peripheral blood lymphocytes as well as tumor infiltrating lymphocytes (TILs) with significant clinical results. Despite these successes, the limitations of the current strategies are also emerging and novel approaches are needed. The bone marrow (BM) is an immunological niche that houses T cells with specificity for previously encountered antigens, including tumor-associated antigens from certain solid cancers. This study sought to improve our understanding of tumor-specific BM T cells in the context of solid tumors by comparing them with TILs, and to assess whether there is a rationale for using the BM as a source of T cells for ACT against solid malignancies. Herein, we demonstrate that T cells from the BM appear superior to TILs as a source of cells for cellular therapy. Specifically, they possess a memory-enriched phenotype and exhibit improved effector function, greater persistence within a tumor-bearing host, and the capacity for increased tumor infiltration. Taken together, these data provide a foundation for further exploring the BM as a source of tumor-specific T cells for ACT in solid malignancies.

Mechanistic target of rapamycin (mTOR) regulates self-sustained quiescence, tumor indolence, and late clinical metastasis in a Beclin-1-dependent manner.

Self-sustained quiescence (SSQ) has been characterized as a stable but reversible non-proliferative cellular state that limits the cloning of cultured cancer cells. By developing refined clonogenic assays, we showed here that cancer cells in SSQ can be selected with anticancer agents and that culture at low cell density induced SSQ in pancreas and prostate adenocarcinoma cells. Pre-culture of cells in 3D or their pretreatment with pharmacological inhibitors of mechanistic target of rapamycin (mTOR) synergize with low cell density for induction of SSQ in a Beclin-1-dependent manner. Dissociated pancreatic adenocarcinoma (PAAD) cells rendered defective for SSQ by down-regulating Beclin-1 expression exhibit higher tumor growth rate when injected subcutaneously into mice. Conversely, dissociated PAAD cells in SSQ promote the formation of small indolent tumors that eventually transitioned to a rapid growth phase. Ex vivo clonogenic assays showed that up to 40% of clonogenic cancer cells enzymatically dissociated from resected fast-growing tumors could enter SSQ, suggesting that SSQ could significantly impact the proliferation of cancer cells that are naturally dispersed from tumors. Remarkably, the kinetics of clinical metastatic recurrence in 124 patients with pancreatic adenocarcinoma included in the TGCA-PAAD project could be predicted from Beclin-1 and Cyclin-A2 mRNA levels in their primary tumor, Cyclin A2 mRNA being a marker of both cell proliferation and mTOR complex 1 activity. Overall, our data show that SSQ is likely to promote the late development of clinical metastases and suggest that identifying new agents targeting cancer cells in SSQ could help improve patient survival.

Empowering the Potential of CAR-T Cell Immunotherapies by Epigenetic Reprogramming.

T-cell-based, personalized immunotherapy can nowadays be considered the mainstream treatment for certain blood cancers, with a high potential for expanding indications. Chimeric antigen receptor T cells (CAR-Ts), an ex vivo genetically modified T-cell therapy product redirected to target an antigen of interest, have achieved unforeseen successes in patients with B-cell hematologic malignancies. Frequently, however, CAR-T cell therapies fail to provide durable responses while they have met with only limited success in treating solid cancers because unique, unaddressed challenges, including poor persistence, impaired trafficking to the tumor, and site penetration through a hostile microenvironment, impede their efficacy. Increasing evidence suggests that CAR-Ts' in vivo performance is associated with T-cell intrinsic features that may be epigenetically altered or dysregulated. In this review, we focus on the impact of epigenetic regulation on T-cell differentiation, exhaustion, and tumor infiltration and discuss how epigenetic reprogramming may enhance CAR-Ts' memory phenotype, trafficking, and fitness, contributing to the development of a new generation of potent CAR-T immunotherapies.

Persistence of intramyocardially transplanted murine induced pluripotent stem cell-derived cardiomyocytes from different developmental stages.

BACKGROUND: Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are regarded as promising cell type for cardiac cell replacement therapy, but it is not known whether the developmental stage influences their persistence and functional integration in the host tissue, which are crucial for a long-term therapeutic benefit. To investigate this, we first tested the cell adhesion capability of murine iPSC-CM in vitro at three different time points during the differentiation process and then examined cell persistence and quality of electrical integration in the infarcted myocardium in vivo. METHODS: To test cell adhesion capabilities in vitro, iPSC-CM were seeded on fibronectin-coated cell culture dishes and decellularized ventricular extracellular matrix (ECM) scaffolds. After fixed periods of time, stably attached cells were quantified. For in vivo experiments, murine iPSC-CM expressing enhanced green fluorescent protein was injected into infarcted hearts of adult mice. After 6-7 days, viable ventricular tissue slices were prepared to enable action potential (AP) recordings in transplanted iPSC-CM and surrounding host cardiomyocytes. Afterwards, slices were lysed, and genomic DNA was prepared, which was then used for quantitative real-time PCR to evaluate grafted iPSC-CM count. RESULTS: The in vitro results indicated differences in cell adhesion capabilities between day 14, day 16, and day 18 iPSC-CM with day 14 iPSC-CM showing the largest number of attached cells on ECM scaffolds. After intramyocardial injection, day 14 iPSC-CM showed a significant higher cell count compared to day 16 iPSC-CM. AP measurements revealed no significant difference in the quality of electrical integration and only minor differences in AP properties between d14 and d16 iPSC-CM. CONCLUSION: The results of the present study demonstrate that the developmental stage at the time of transplantation is crucial for the persistence of transplanted iPSC-CM. iPSC-CM at day 14 of differentiation showed the highest persistence after transplantation in vivo, which may be explained by a higher capability to adhere to the extracellular matrix.

Targeting Cbx3/HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence.

Immune checkpoint blockade (ICB) relieves CD8+ T-cell exhaustion in most mutated tumors, and TCF-1 is implicated in converting progenitor exhausted cells to functional effector cells. However, identifying mechanisms that can prevent functional senescence and potentiate CD8+ T-cell persistence for ICB non-responsive and resistant tumors remains elusive. We demonstrate that targeting Cbx3/HP1γ in CD8+ T cells augments transcription initiation and chromatin remodeling leading to increased transcriptional activity at Lef1 and Il21r. LEF-1 and IL-21R are necessary for Cbx3/HP1γ-deficient CD8+ effector T cells to persist and control ovarian cancer, melanoma, and neuroblastoma in preclinical models. The enhanced persistence of Cbx3/HP1γ-deficient CD8+ T cells facilitates remodeling of the tumor chemokine/receptor landscape ensuring their optimal invasion at the expense of CD4+ Tregs. Thus, CD8+ T cells heightened effector function consequent to Cbx3/HP1γ deficiency may be distinct from functional reactivation by ICB, implicating Cbx3/HP1γ as a viable cancer T-cell-based therapy target for ICB resistant, non-responsive solid tumors.

Cytolytic Activity of CAR T Cells and Maintenance of Their CD4+ Subset Is Critical for Optimal Antitumor Activity in Preclinical Solid Tumor Models.

Correlative studies of clinical studies for hematological malignancies have implicated that less differentiated, CD8+-dominant CAR T cell products have greater antitumor activity. Here, we have investigated whether the differentiation status of CAR T cell products affects their antitumor activity in preclinical models of solid tumors. We explored if different activation/expansion protocols, as well as different co-stimulatory domains in the CAR construct, influence the short- and long-term efficacy of CAR T cells against HER2-positive tumors. We generated T cell products that range from the most differentiated (CD28.z; OKT3-antiCD28/RPMI expansion) to the least differentiated (41BB.z; OKT3-RetroNectin/LymphoONE expansion), as judged by cell surface expression of the differentiation markers CCR7 and CD45RA. While the effect of differentiation status was variable with regard to antigen-specific cytokine production, the most differentiated CD28.z CAR T cell products, which were enriched in effector memory T cells, had the greatest target-specific cytolytic activity in vitro. These products also had a greater proliferative capacity and maintained CD4+ T cells upon repeated stimulation in vitro. In vivo, differentiated CD28.z CAR T cells also had the greatest antitumor activity, resulting in complete response. Our results highlight that it is critical to optimize CAR T cell production and that optimal product characteristics might depend on the targeted antigen and/or cancer.

Accurate In-Vivo Quantification of CD19 CAR-T Cells after Treatment with Axicabtagene Ciloleucel (Axi-Cel) and Tisagenlecleucel (Tisa-Cel) Using Digital PCR.

Immunotherapy with CD19-specific chimeric antigen receptor (CAR-) T cells has shown excellent efficacy in relapsed/refractory B-cell cancers. The in vivo expansion and persistence of CAR-T cells after infusion are important response- and toxicity-determining variables, but diagnostic tools are largely missing. We showed previously for axi-cel that digital PCR (dPCR) is excellently suited to monitoring CAR-T cells in vivo. Here, we aimed to develop an analogous dPCR assay for tisa-cel. To do so, we cloned and sequenced the CAR construct from the lentiviral tisa-cel vector and designed primers and Black hole quencher (BHQ) probes complimentary to sequences present in the FMC63 scFv part of axi-cel (assay A), tisa-cel (T), and both constructs (U = "universal"). In conjunction with excellent specificity, all assays have a detection limit of one single CAR copy, corresponding to a sensitivity of approximately 1 in 5000 cells (0.02%) for 100 ng genomic DNA (for one vector copy per transduced cell). The new universal assay was first validated using patient samples previously quantified with the axi-cel-specific dPCR and thereafter applied to quantify and monitor adoptively transferred axi-cel and tisa-cel T cells in post-infusion samples (peripheral blood, bone marrow, liquor, and ascites). Actual CAR-T counts per µl were calculated, taking into account vector copy and peripheral blood mononuclear cell (PBMC) numbers, and showed very good correlation with flow cytometry results. We conclude that our novel dPCR assay is optimally suited to monitoring tisa-cel and axi-cel CAR-T cells in real-time in various body fluids.

Analysis of Antitumor Effects of CAR-T Cells in Mice with Solid Tumors.

Animal models provide an essential tool to study the efficacy of CAR-T cell treatments. Most of the current works test human CAR-T cells in immunodeficient animals, typically NOD Scid Gamma (NSG) mice transplanted with human tumors. Despite the limitations of this model, including the difficulty to study the interaction between CAR-T cells and the human innate system and to assess the toxicity of this therapy, NSG are extensively used for adoptive T cell transfer studies. In this chapter, we will describe the protocols to test CAR-T cells in NSG animals with solid tumors. We first describe the implantation of human xenograft tumors in NSG animals, followed by CAR-T cell administration and assessment of antitumor responses. We will also review the protocols to analyze T cell persistence in the blood of treated animals. Finally, we will focus on the analysis of the tumors at the end point of the experiment, including the percentage, phenotype, and function of tumor infiltrating T cells, and loss of antigen expression.

Adjuvants Enhance the Induction of Germinal Center and Antibody Secreting Cells in Spleen and Their Persistence in Bone Marrow of Neonatal Mice.

Immaturity of the immune system contributes to poor vaccine responses in early life. Germinal center (GC) activation is limited due to poorly developed follicular dendritic cells (FDC), causing generation of few antibody-secreting cells (ASCs) with limited survival and transient antibody responses. Herein, we compared the potential of five adjuvants, namely LT-K63, mmCT, MF59, IC31, and alum to overcome limitations of the neonatal immune system and to enhance and prolong responses of neonatal mice to a pneumococcal conjugate vaccine Pnc1-TT. The adjuvants LT-K63, mmCT, MF59, and IC31 significantly enhanced GC formation and FDC maturation in neonatal mice when co-administered with Pnc1-TT. This enhanced GC induction correlated with significantly enhanced vaccine-specific ASCs by LT-K63, mmCT, and MF59 in spleen 14 days after immunization. Furthermore, mmCT, MF59, and IC31 prolonged the induction of vaccine-specific ASCs in spleen and increased their persistence in bone marrow up to 9 weeks after immunization, as previously shown for LT-K63. Accordingly, serum Abs persisted above protective levels against pneumococcal bacteremia and pneumonia. In contrast, alum only enhanced the primary induction of vaccine-specific IgG Abs, which was transient. Our comparative study demonstrated that, in contrast to alum, LT-K63, mmCT, MF59, and IC31 can overcome limitations of the neonatal immune system and enhance both induction and persistence of protective immune response when administered with Pnc1-TT. These adjuvants are promising candidates for early life vaccination.

Elevation and persistence of CD8 T-cells in HIV infection: the Achilles heel in the ART era.

INTRODUCTION: HIV infection leads to a disturbed T-cell homeostasis, featured by a depletion of CD4 T-cells and a persistent elevation of CD8 T-cells over disease progression. Most effort of managing HIV infection has been focused on CD4 T-cell recovery, while changes in the CD8 compartment were relatively underappreciated in the past. METHODS: A comprehensive literature review of publications in English language was conducted using major electronic databases. Our search was focused on factors contributing to CD8 T-cell dynamics in HIV infection and following antiretroviral therapy (ART). DISCUSSION: Normalization of CD8 counts is seldom observed even with optimal CD4 recovery following long-term treatment. Initiation of ART in primary HIV infection leads to enhanced normalization of CD8 count compared with long-term ART initiated in chronic infection. Importantly, such CD8 elevation in treated HIV infection is associated with an increased risk of inflammatory non-AIDS-related clinical events independent of CD4 T-cell recovery. The mechanisms underlying CD8 persistence remain largely unknown, which may include bystander activation, exhaustion and immunosenescence of CD8 T-cells. The information provided herein will lead to a better understanding of factors associated with CD8 persistence and contribute to the development of strategies aiming at CD8 normalization. CONCLUSIONS: Persistence of CD8 T-cell elevation in treated HIV-infected patients is associated with an increased risk of non-AIDS-related events. Now that advances in ART have led to decreased AIDS-related opportunistic diseases, more attention has been focused on reducing non-AIDS events and normalizing persistent CD8 T-cell elevation.

T Cell Maturation Stage Prior to and During GMP Processing Informs on CAR T Cell Expansion in Patients.

Autologous T cells were genetically modified to express a chimeric antigen receptor (CAR) directed toward carboxy-anhydrase-IX (CAIX) and used to treat patients with CAIX-positive metastatic renal cell carcinoma. In this study, we questioned whether the T cell maturation stage in the pre-infusion product affected CAIX CAR expression and function in vitro as well as in vivo CAR T cell numbers and expansion. During the 14 days expansion of CAR T cells prior to administration, we observed shifts from a predominant CD4 to a CD8 T cell phenotype and from a significant fraction of naïve to central effector T cells. Surface expression of the CAR was equally distributed among different T cell subsets and T cell maturation stages. During T cell culture days 14-18 (which covered patient treatment days 1-5), T cells demonstrated a decline in CAR expression level per cell irrespective of T cell maturation stage, although the proportion of CAR-positive T cells and CAR-mediated T cell effector functions remained similar for both CD4 and CD8 T cell populations. Notably, patients with a higher fraction of naïve CD8 T cells at baseline (prior to genetic modification) or central effector CD8 T cells at 2 weeks of CAR T cell culture demonstrated a higher fold expansion and absolute numbers of circulating CAR T cells at 1 month after start of therapy. We conclude that the T cell maturation stage prior to and during CAR T cell expansion culture is related to in vivo CAR T cell expansion.