HIV is adept at avoiding naturally generated T cell responses; therefore, there is a need to develop HIV-specific T cells with greater potency for use in HIV cure strategies. Starting with a ...CD4-based chimeric antigen receptor (CAR) that was previously used without toxicity in clinical trials, we optimized the vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains to determine which components augmented the ability of T cells to control HIV replication. This re-engineered CAR was at least 50-fold more potent in vitro at controlling HIV replication than the original CD4 CAR, or a TCR-based approach, and substantially better than broadly neutralizing antibody-based CARs. A humanized mouse model of HIV infection demonstrated that T cells expressing optimized CARs were superior at expanding in response to antigen, protecting CD4 T cells from infection, and reducing viral loads compared to T cells expressing the original, clinical trial CAR. Moreover, in a humanized mouse model of HIV treatment, CD4 CAR T cells containing the 4-1BB costimulatory domain controlled HIV spread after ART removal better than analogous CAR T cells containing the CD28 costimulatory domain. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and that CAR T cells could be important components of an HIV cure strategy.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR T cell exhaustion in the solid ...tumor microenvironment. To study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of T cell exhaustion and discover, both in vitro and in CAR T cell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR T cell exhaustion. Our findings shed light on the plasticity of human CAR T cells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR T cell therapy in solid tumors by preventing or delaying CAR T cell dysfunction.
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•CAR T cells under chronic antigen stimulation show hallmarks of T cell exhaustion•CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition•CAR T cells with NK-like transition were identified in patients after treatment•Unlike WT CAR T cells, ID3 and SOX4 knockout CAR T cells retain anti-tumor immunity
Continuous antigen exposure drives CAR T cell exhaustion and promotes CD8+ T-to-NK-like T cell transition. Transcription factors ID3 and SOX4 are upregulated during CAR dysfunction and regulate genes associated with exhaustion, including NK receptors. Knocking out ID3 and SOX4 in CAR T cells slows dysfunction and improves anti-tumor immunity.
BackgroundWe conducted a phase I clinical trial that infused CCR5 gene-edited CD4+ T cells to determine how these T cells can better enable HIV cure strategies.MethodsThe aim of trial was to develop ...RNA-based approaches to deliver zinc finger nuclease (ZFN), evaluate the effect of CCR5 gene-edited CD4+ T cells on the HIV-specific T cell response, test the ability of infused CCR5 gene-edited T cells to delay viral rebound during analytical treatment interruption, and determine whether individuals heterozygous for CCR5 Δ32 preferentially benefit. We enrolled 14 individuals living with HIV whose viral load was well controlled by antiretroviral therapy (ART). We measured the time to viral rebound after ART withdrawal, the persistence of CCR5-edited CD4+ T cells, and whether infusion of 10 billion CCR5-edited CD4+ T cells augmented the HIV-specific immune response.ResultsInfusion of the CD4+ T cells was well tolerated, with no serious adverse events. We observed a modest delay in the time to viral rebound relative to historical controls; however, 3 of the 14 individuals, 2 of whom were heterozygous for CCR5 Δ32, showed post-viral rebound control of viremia, before ultimately losing control of viral replication. Interestingly, only these individuals had substantial restoration of HIV-specific CD8+ T cell responses. We observed immune escape for 1 of these reinvigorated responses at viral recrudescence, illustrating a direct link between viral control and enhanced CD8+ T cell responses.ConclusionThese findings demonstrate how CCR5 gene-edited CD4+ T cell infusion could aid HIV cure strategies by augmenting preexisting HIV-specific immune responses.REGISTRATIONClinicalTrials.gov NCT02388594.FundingNIH funding (R01AI104400, UM1AI126620, U19AI149680, T32AI007632) was provided by the National Institute of Allergy and Infectious Diseases (NIAID), the National Institute on Drug Abuse (NIDA), the National Institute of Mental Health (NIMH), and the National Institute of Neurological Disorders and Stroke (NINDS). Sangamo Therapeutics also provided funding for these studies.
Abstract Based on the success in animal models of type 1 diabetes (T1D), clinical trials of adoptive regulatory T cell (Treg) therapy are underway using ex vivo expanded polyclonal Tregs. However, ...pre-clinical data also demonstrate that islet-specific Tregs are more potent than polyclonal Tregs at reversing T1D. Translation of this approach into man will require methods to generate large populations of islet-specific Tregs which, to date, has proved to be a major hurdle. Here we demonstrate the feasibility of lentiviral-mediated T cell receptor (TCR) gene transfer to confer antigen specificity on polyclonal human Tregs. Targeting has been achieved using TCRs isolated from human islet-specific and viral-specific CD4+ T cell clones. Engineered T cells demonstrated expression of ectopically-delivered TCRs, resulting in endowment of cognate antigen-specific responses. This enabled antigen-specific suppression at increased potency compared to polyclonal Tregs. However, cells transduced with islet-specific TCRs were less responsive to cognate antigen than viral-specific TCRs, and in some cases, required additional methods to isolate functional antigen-specific Tregs. This study demonstrates the potential of TCR gene transfer to develop islet-specific Treg therapies for effective treatment of T1D, but also highlights that additional optimisation may be required to achieve its full potential.
HIV-1 entry can be inhibited by soluble peptides from the gp41 heptad repeat-2 (HR2) domain that interfere with formation of the 6-helix bundle during fusion. Inhibition has also been seen when these ...peptides are conjugated to anchoring molecules and over-expressed on the cell surface. We hypothesized that potent anti-HIV activity could be achieved if a 34 amino acid peptide from HR2 (C34) were brought to the site of virus-cell interactions by conjugation to the amino termini of HIV-1 coreceptors CCR5 or CXCR4. C34-conjugated coreceptors were expressed on the surface of T cell lines and primary CD4 T cells, retained the ability to mediate chemotaxis in response to cognate chemokines, and were highly resistant to HIV-1 utilization for entry. Notably, C34-conjugated CCR5 and CXCR4 each exhibited potent and broad inhibition of HIV-1 isolates from diverse clades irrespective of tropism (i.e., each could inhibit R5, X4 and dual-tropic isolates). This inhibition was highly specific and dependent on positioning of the peptide, as HIV-1 infection was poorly inhibited when C34 was conjugated to the amino terminus of CD4. C34-conjugated coreceptors could also inhibit HIV-1 isolates that were resistant to the soluble HR2 peptide inhibitor, enfuvirtide. When introduced into primary cells, CD4 T cells expressing C34-conjugated coreceptors exhibited physiologic responses to T cell activation while inhibiting diverse HIV-1 isolates, and cells containing C34-conjugated CXCR4 expanded during HIV-1 infection in vitro and in a humanized mouse model. Notably, the C34-conjugated peptide exerted greater HIV-1 inhibition when conjugated to CXCR4 than to CCR5. Thus, antiviral effects of HR2 peptides can be specifically directed to the site of viral entry where they provide potent and broad inhibition of HIV-1. This approach to engineer HIV-1 resistance in functional CD4 T cells may provide a novel cell-based therapeutic for controlling HIV infection in humans.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rhesus (rh) but not human (hu) TRIM5α potently restricts human immunodeficiency virus (HIV)-1 infection. It is not clear why huTRIM5α fails to effectively block HIV infection, but it is thought to ...have a lower affinity for the viral core. Using primary human CD4 T cells, we investigated the ability of huTRIM5α, rhTRIM5α, and the huTRIM5αR323–332 B30.2/SPRY patch-mutant to form cytoplasmic bodies, postulated as key components of the HIV-1 restriction apparatus. Both rhTRIM5α and huTRIM5αR323–332 formed pronounced cytoplasmic bodies, whereas cytoplasmic bodies in T cells overexpressing huTRIM5α were present but more difficult to detect. As expression of all three TRIM5α orthologs was similar at the RNA level, we next investigated the role of protein stability in conferring TRIM5α-mediated HIV-1 restriction. Both steady-state and pulse-chase experiments revealed that the huTRIM5α protein was much less stable than rhTRIM5α, and this difference correlated with higher self-ubiquitination activity. Using a stabilized form of huTRIM5α in which the steady-state expression level was more similar to rhTRIM5α, we observed comparable HIV-1 restriction activity in multi-round HIV-1 challenge assays. Lastly, primary human CD4 T cells expressing a stabilized huTRIM5α were protected from HIV-1-mediated destruction in vivo, indicating that efforts to stabilize huTRIM5α should have significant long-term therapeutic value.
In carcinogen-driven cancers, a high mutational burden results in neoepitopes that can be recognized immunologically. Such carcinogen-induced tumors may evade this immune response through ..."immunoediting," whereby tumors adapt to immune pressure and escape T cell-mediated killing. Many tumors lack a high neoepitope burden, and it remains unclear whether immunoediting occurs in such cases. Here, we evaluated T cell immunity in an autochthonous mouse model of pancreatic cancer and found a low mutational burden, absence of predicted neoepitopes derived from tumor mutations, and resistance to checkpoint immunotherapy. Spontaneous tumor progression was identical in the presence or absence of T cells. Moreover, tumors arising in T cell-depleted mice grew unchecked in immune-competent hosts. However, introduction of the neoantigen ovalbumin (OVA) led to tumor rejection and T cell memory, but this did not occur in OVA immune-tolerant mice. Thus, immunoediting does not occur in this mouse model - a likely consequence, not a cause, of absent neoepitopes. Because many human tumors also have a low missense mutational load and minimal neoepitope burden, our findings have clinical implications for the design of immunotherapy for patients with such tumors.
CCR5, a cell surface molecule critical for the transmission and spread of HIV-1, is dynamically regulated during T cell activation and differentiation. The molecular mechanism linking T cell ...activation to modulation of CCR5 expression remains undefined. Kruppel-like factor 2 (KLF2) is a transcription factor that promotes quiescence, survival, and in part by modulating chemokine receptor levels, induces homing to secondary lymphoid organs. Given the relationship between T cell activation and chemokine receptor expression, we tested whether the abundance of KLF2 after T cell activation regulates CCR5 expression and, thus, susceptibility of a T cell to CCR5-dependent HIV-1 strains (R5). We observed a strong correlation between T cell activation, expression of KLF2 and CCR5, and susceptibility to infection. To directly measure how KLF2 affects CCR5 regulation, we introduced small interfering RNA targeting KLF2 expression and demonstrated that reduced KLF2 expression also resulted in less CCR5. Chromatin immunoprecipitation assays identified KLF2 bound to the CCR5 promoter in resting but not CD3/28 activated T cells, suggesting that KLF2 directly regulates CCR5 expression. Introduction of KLF2 under control of a heterologous promoter could restore CCR5 expression and R5 susceptibility to CD3/28 costimulated T cells and some transformed cell lines. Thus, KLF2 is a host factor that modulates CCR5 expression in CD4 T cells and influences susceptibility to R5 infection.
Overcrowding has been reported in Emergency Medicine literature for decades and has demonstrated a negative impact on adverse events including mortality, quality of care, elopement, return to the ...emergency department (ED), medical errors, and timeliness of patient care 1-3. ...of ED volumes exceeding ED bed capacity, patients are being cared for in hallways outside of conventional rooms. Limitations included studying only voluntarily completed and returned surveys of discharged ED patients, most of whom were female, at a single site and variables such as length of hallway bed stay or when order of hallway bed experience took place were not determined.
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