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1.
Immunity ; 55(7): 1200-1215.e6, 2022 07 12.
Article in English | MEDLINE | ID: mdl-35637103

ABSTRACT

Soon after activation, CD4+ T cells are segregated into BCL6+ follicular helper (Tfh) and BCL6- effector (Teff) T cells. Here, we explored how these subsets are maintained during chronic antigen stimulation using the mouse chronic LCMV infection model. Using single cell-transcriptomic and epigenomic analyses, we identified a population of PD-1+ TCF-1+ CD4+ T cells with memory-like features. TCR clonal tracing and adoptive transfer experiments demonstrated that these cells have self-renewal capacity and continue to give rise to both Teff and Tfh cells, thus functioning as progenitor cells. Conditional deletion experiments showed Bcl6-dependent development of these progenitors, which were essential for sustaining antigen-specific CD4+ T cell responses to chronic infection. An analogous CD4+ T cell population developed in draining lymph nodes in response to tumors. Our study reveals the heterogeneity and plasticity of CD4+ T cells during persistent antigen exposure and highlights their population dynamics through a stable, bipotent intermediate state.


Subject(s)
Antigens , T-Lymphocytes, Helper-Inducer , Adoptive Transfer , Animals , Cell Differentiation , Mice , Proto-Oncogene Proteins c-bcl-6/genetics , Stem Cells
2.
Mol Cell ; 72(4): 636-649.e8, 2018 11 15.
Article in English | MEDLINE | ID: mdl-30293785

ABSTRACT

Class switch recombination (CSR) is a DNA recombination reaction that diversifies the effector component of antibody responses. CSR is initiated by activation-induced cytidine deaminase (AID), which targets transcriptionally active immunoglobulin heavy chain (Igh) switch donor and acceptor DNA. The 3' Igh super-enhancer, 3' regulatory region (3'RR), is essential for acceptor region transcription, but how this function is regulated is unknown. Here, we identify the chromatin reader ZMYND8 as an essential regulator of the 3'RR. In B cells, ZMYND8 binds promoters and super-enhancers, including the Igh enhancers. ZMYND8 controls the 3'RR activity by modulating the enhancer transcriptional status. In its absence, there is increased 3'RR polymerase loading and decreased acceptor region transcription and CSR. In addition to CSR, ZMYND8 deficiency impairs somatic hypermutation (SHM) of Igh, which is also dependent on the 3'RR. Thus, ZMYND8 controls Igh diversification in mature B lymphocytes by regulating the activity of the 3' Igh super-enhancer.


Subject(s)
Chromatin Assembly and Disassembly/genetics , Immunoglobulin Class Switching/genetics , Immunoglobulin Heavy Chains/genetics , Tumor Suppressor Proteins/genetics , Animals , B-Lymphocytes , Cell Line , Chromatin/genetics , Chromatin/metabolism , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , DNA/genetics , Enhancer Elements, Genetic , Gene Rearrangement , Humans , MYND Domains , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Somatic Hypermutation, Immunoglobulin/genetics , Tumor Suppressor Proteins/metabolism
3.
Nat Methods ; 18(8): 881-892, 2021 08.
Article in English | MEDLINE | ID: mdl-34282327

ABSTRACT

T cells express T cell receptors (TCRs) composed of somatically recombined TCRα and TCRß chains, which mediate recognition of major histocompatibility complex (MHC)-antigen complexes and drive the antigen-specific adaptive immune response to pathogens and cancer. The TCR repertoire in each individual is highly diverse, which allows for recognition of a wide array of foreign antigens, but also presents a challenge in analyzing this response using conventional methods. Recent studies have developed high-throughput sequencing technologies to identify TCR sequences, analyze their antigen specificities using experimental and computational tools, and pair TCRs with transcriptional and epigenetic cell state phenotypes in single cells. In this Review, we highlight these technological advances and describe how they have been applied to discover fundamental insights into T cell-mediated immunity.


Subject(s)
Antigens/immunology , High-Throughput Nucleotide Sequencing/methods , Major Histocompatibility Complex/immunology , Receptors, Antigen, T-Cell/immunology , Single-Cell Analysis/methods , T-Lymphocytes/immunology , Adaptive Immunity , Animals , Humans
4.
Nature ; 561(7724): 479-484, 2018 09.
Article in English | MEDLINE | ID: mdl-30258136

ABSTRACT

Individuals infected with HIV-1 require lifelong antiretroviral therapy, because interruption of treatment leads to rapid rebound viraemia. Here we report on a phase 1b clinical trial in which a combination of 3BNC117 and 10-1074, two potent monoclonal anti-HIV-1 broadly neutralizing antibodies that target independent sites on the HIV-1 envelope spike, was administered during analytical treatment interruption. Participants received three infusions of 30 mg kg-1 of each antibody at 0, 3 and 6 weeks. Infusions of the two antibodies were generally well-tolerated. The nine enrolled individuals with antibody-sensitive latent viral reservoirs maintained suppression for between 15 and more than 30 weeks (median of 21 weeks), and none developed viruses that were resistant to both antibodies. We conclude that the combination of the anti-HIV-1 monoclonal antibodies 3BNC117 and 10-1074 can maintain long-term suppression in the absence of antiretroviral therapy in individuals with antibody-sensitive viral reservoirs.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/therapeutic use , HIV Antibodies/therapeutic use , HIV Infections/drug therapy , HIV Infections/immunology , HIV-1/immunology , Virus Latency/immunology , Adolescent , Adult , Aged , Anti-HIV Agents/administration & dosage , Anti-HIV Agents/immunology , Anti-HIV Agents/therapeutic use , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Humanized , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/adverse effects , Antibodies, Neutralizing/immunology , Binding Sites, Antibody , Broadly Neutralizing Antibodies , Carrier State/drug therapy , Carrier State/immunology , Carrier State/virology , Drug Combinations , Drug Resistance, Viral , Female , HIV Antibodies/administration & dosage , HIV Antibodies/adverse effects , HIV Antibodies/immunology , HIV Envelope Protein gp160/immunology , HIV Infections/virology , HIV-1/isolation & purification , Historically Controlled Study , Humans , Infusions, Intravenous , Male , Middle Aged , Phylogeny , Viremia/drug therapy , Viremia/immunology , Viremia/prevention & control , Viremia/virology , Virus Activation/immunology , Young Adult
5.
Proc Natl Acad Sci U S A ; 115(48): E11341-E11348, 2018 11 27.
Article in English | MEDLINE | ID: mdl-30420517

ABSTRACT

Combination antiretroviral therapy controls but does not cure HIV-1 infection because a small fraction of cells harbor latent viruses that can produce rebound viremia when therapy is interrupted. The circulating latent virus reservoir has been documented by a variety of methods, most prominently by viral outgrowth assays (VOAs) in which CD4+ T cells are activated to produce virus in vitro, or more recently by amplifying proviral near full-length (NFL) sequences from DNA. Analysis of samples obtained in clinical studies in which individuals underwent analytical treatment interruption (ATI), showed little if any overlap between circulating latent viruses obtained from outgrowth cultures and rebound viruses from plasma. To determine whether intact proviruses amplified from DNA are more closely related to rebound viruses than those obtained from VOAs, we assayed 12 individuals who underwent ATI after infusion of a combination of two monoclonal anti-HIV-1 antibodies. A total of 435 intact proviruses obtained by NFL sequencing were compared with 650 latent viruses from VOAs and 246 plasma rebound viruses. Although, intact NFL and outgrowth culture sequences showed similar levels of stability and diversity with 39% overlap, the size of the reservoir estimated from NFL sequencing was larger than and did not correlate with VOAs. Finally, intact proviruses documented by NFL sequencing showed no sequence overlap with rebound viruses; however, they appear to contribute to recombinant viruses found in plasma during rebound.


Subject(s)
CD4-Positive T-Lymphocytes/virology , HIV Infections/virology , HIV-1/physiology , Proviruses/physiology , Anti-HIV Agents/administration & dosage , Antibodies, Monoclonal, Humanized , Antibodies, Neutralizing/administration & dosage , Broadly Neutralizing Antibodies , HIV Antibodies/administration & dosage , HIV Infections/drug therapy , HIV-1/classification , HIV-1/genetics , HIV-1/growth & development , Humans , Phylogeny , Proviruses/classification , Proviruses/genetics , Proviruses/growth & development , Virus Latency , Virus Replication
6.
J Virol ; 93(8)2019 04 15.
Article in English | MEDLINE | ID: mdl-30700598

ABSTRACT

The role of lymphoid tissue as a potential source of HIV-1 rebound following interruption of antiretroviral therapy (ART) is uncertain. To address this issue, we compared the latent viruses obtained from CD4+ T cells in peripheral blood and lymph nodes to viruses emerging during treatment interruption. Latent viruses were characterized by sequencing near-full-length (NFL) proviral DNA and env from viral outgrowth assays (VOAs). Five HIV-1-infected individuals on ART were studied, four of whom participated in a clinical trial of a TLR9 agonist that included an analytical treatment interruption. We found that 98% of intact or replication-competent clonal sequences overlapped between blood and lymph node. In contrast, there was no overlap between 205 latent reservoir and 125 rebound sequences in the four individuals who underwent treatment interruption. However, rebound viruses could be accounted for by recombination. The data suggest that CD4+ T cells carrying latent viruses circulate between blood and lymphoid tissues in individuals on ART and support the idea that recombination may play a role in the emergence of rebound viremia.IMPORTANCE HIV-1 persists as a latent infection in CD4+ T cells that can be found in lymphoid tissues in infected individuals during ART. However, the importance of this tissue reservoir and its contribution to viral rebound upon ART interruption are not clear. In this study, we sought to compare latent HIV-1 from blood and lymph node CD4+ T cells from five HIV-1-infected individuals. Further, we analyzed the contribution of lymph node viruses to viral rebound. We observed that the frequencies of intact proviruses were the same in blood and lymph node. Moreover, expanded clones of T cells bearing identical proviruses were found in blood and lymph node. These latent reservoir sequences did not appear to be the direct origin of rebound virus. Instead, latent proviruses were found to contribute to the rebound compartment by recombination.


Subject(s)
Anti-Retroviral Agents/administration & dosage , CD4-Positive T-Lymphocytes , DNA, Viral/blood , HIV Infections , HIV-1/metabolism , Lymph Nodes , Proviruses/metabolism , Adult , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Female , HIV Infections/blood , HIV Infections/drug therapy , Humans , Lymph Nodes/metabolism , Lymph Nodes/virology , Male , Middle Aged , Toll-Like Receptor 9/agonists , Toll-Like Receptor 9/blood
7.
bioRxiv ; 2023 Nov 29.
Article in English | MEDLINE | ID: mdl-38077050

ABSTRACT

Decreased intra-tumor heterogeneity (ITH) correlates with increased patient survival and immunotherapy response. However, even highly homogenous tumors may display variability in their aggressiveness, and how immunologic-factors impinge on their aggressiveness remains understudied. Here we studied the mechanisms responsible for the immune-escape of murine tumors with low ITH. We compared the temporal growth of homogeneous, genetically-similar single-cell clones that are rejected vs. those that are not-rejected after transplantation in-vivo using single-cell RNA sequencing and immunophenotyping. Non-rejected clones showed high infiltration of tumor-associated-macrophages (TAMs), lower T-cell infiltration, and increased T-cell exhaustion compared to rejected clones. Comparative analysis of rejection-associated gene expression programs, combined with in-vivo CRISPR knockout screens of candidate mediators, identified Mif (macrophage migration inhibitory factor) as a regulator of immune rejection. Mif knockout led to smaller tumors and reversed non-rejection-associated immune composition, particularly, leading to the reduction of immunosuppressive macrophage infiltration. Finally, we validated these results in melanoma patient data.

8.
Cancer Cell ; 41(4): 776-790.e7, 2023 04 10.
Article in English | MEDLINE | ID: mdl-37001526

ABSTRACT

Paired single-cell RNA and T cell receptor sequencing (scRNA/TCR-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scRNA/TCR-seq analysis of 187,650 T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients with non-small cell lung cancer after immune checkpoint blockade (ICB). Regions with viable cancer cells are enriched for exhausted CD8+ T cells, regulatory CD4+ T cells (Treg), and follicular helper CD4+ T cells (TFH). Tracking T cell clonotypes across tissues, combined with neoantigen specificity assays, reveals that TFH and tumor-specific exhausted CD8+ T cells are clonally linked to TCF7+SELL+ progenitors in tumor draining LNs, and progressive exhaustion trajectories of CD8+ T, Treg, and TFH cells with proximity to the tumor microenvironment. Finally, longitudinal tracking of tumor-specific CD8+ and CD4+ T cell clones reveals persistence in the peripheral blood for years after ICB therapy.


Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , CD8-Positive T-Lymphocytes , Carcinoma, Non-Small-Cell Lung/drug therapy , Immune Checkpoint Inhibitors/therapeutic use , Lung Neoplasms/drug therapy , Receptors, Antigen, T-Cell , Clone Cells , Tumor Microenvironment
9.
J Clin Invest ; 131(20)2021 10 15.
Article in English | MEDLINE | ID: mdl-34651586

ABSTRACT

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-the-shelf" precision immunotherapies, alleviating limitations of personalized treatments.


Subject(s)
Antigens, Neoplasm/immunology , Melanoma/immunology , ras Proteins/immunology , Cell Line, Tumor , HLA-A Antigens/immunology , Humans , Lymphocytes, Tumor-Infiltrating/immunology , Receptors, Antigen, T-Cell/immunology , ras Proteins/genetics
10.
J Exp Med ; 216(10): 2253-2264, 2019 10 07.
Article in English | MEDLINE | ID: mdl-31350309

ABSTRACT

HIV-1 infection requires lifelong therapy with antiretroviral drugs due to the existence of a latent reservoir of transcriptionally inactive integrated proviruses. The goal of HIV-1 cure research is to eliminate or functionally silence this reservoir. To this end, there are numerous ongoing studies to evaluate immunological approaches, including monoclonal antibody therapies. Evaluating the results of these studies requires sensitive and specific measures of the reservoir. Here, we describe a relatively high-throughput combined quantitative PCR (qPCR) and next-generation sequencing method. Four different qPCR probes covering the packaging signal (PS), group-specific antigen (gag), polymerase (pol), and envelope (env) are combined in a single multiplex reaction to detect the HIV-1 genome in limiting dilution samples followed by sequence verification of individual reactions that are positive for combinations of any two of the four probes (Q4PCR). This sensitive and specific approach allows for an unbiased characterization of the HIV-1 latent reservoir.


Subject(s)
Genome, Viral , HIV Infections/genetics , HIV-1/genetics , High-Throughput Nucleotide Sequencing , Real-Time Polymerase Chain Reaction , Viral Proteins/genetics , Anti-Retroviral Agents/administration & dosage , Female , HIV Infections/drug therapy , Humans , Male
11.
J Exp Med ; 216(10): 2316-2330, 2019 10 07.
Article in English | MEDLINE | ID: mdl-31345931

ABSTRACT

Human anti-HIV-1 broadly neutralizing antibodies (bNAbs) protect against infection in animal models. However, bNAbs have not been elicited by vaccination in diverse wild-type animals or humans, in part because B cells expressing the precursors of these antibodies do not recognize most HIV-1 envelopes (Envs). Immunogens have been designed that activate these B cell precursors in vivo, but they also activate competing off-target responses. Here we report on a complementary approach to expand specific B cells using an anti-idiotypic antibody, iv8, that selects for naive human B cells expressing immunoglobulin light chains with 5-amino acid complementarity determining region 3s, a key feature of anti-CD4 binding site (CD4bs)-specific VRC01-class antibodies. In mice, iv8 induced target cells to expand and mature in the context of a polyclonal immune system and produced serologic responses targeting the CD4bs on Env. In summary, the results demonstrate that an anti-idiotypic antibody can specifically recognize and expand rare B cells that express VRC01-class antibodies against HIV-1.


Subject(s)
Antibodies, Anti-Idiotypic/immunology , Antibodies, Neutralizing/immunology , B-Lymphocytes/immunology , HIV Antibodies/immunology , HIV Envelope Protein gp160/immunology , HIV Infections/immunology , HIV-1/immunology , Animals , HIV Envelope Protein gp160/genetics , HIV Infections/genetics , Humans , Mice , Mice, Transgenic
12.
Nat Med ; 24(5): 604-609, 2018 05.
Article in English | MEDLINE | ID: mdl-29686423

ABSTRACT

Despite suppressive combination antiretroviral therapy (ART), latent HIV-1 proviruses persist in patients. This latent reservoir is established within 48-72 h after infection, has a long half-life1,2, enables viral rebound when ART is interrupted, and is the major barrier to a cure for HIV-1 3 . Latent cells are exceedingly rare in blood (∼1 per 1 × 106 CD4+ T cells) and are typically enumerated by indirect means, such as viral outgrowth assays4,5. We report a new strategy to purify and characterize single reactivated latent cells from HIV-1-infected individuals on suppressive ART. Surface expression of viral envelope protein was used to enrich reactivated latent T cells producing HIV RNA, and single-cell analysis was performed to identify intact virus. Reactivated latent cells produce full-length viruses that are identical to those found in viral outgrowth cultures and represent clones of in vivo expanded T cells, as determined by their T cell receptor sequence. Gene-expression analysis revealed that these cells share a transcriptional profile that includes expression of genes implicated in silencing the virus. We conclude that reactivated latent T cells isolated from blood can share a gene-expression program that allows for cell division without activation of the cell death pathways that are normally triggered by HIV-1 replication.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Gene Expression Profiling , HIV-1/physiology , Virus Latency/physiology , Clone Cells , Humans , Principal Component Analysis , RNA, Viral/metabolism , Sequence Analysis, RNA , Single-Cell Analysis
13.
Nat Med ; 24(11): 1701-1707, 2018 11.
Article in English | MEDLINE | ID: mdl-30258217

ABSTRACT

Monotherapy of HIV-1 infection with single antiretroviral agents is ineffective because error-prone HIV-1 replication leads to the production of drug-resistant viral variants1,2. Combinations of drugs can establish long-term control, however, antiretroviral therapy (ART) requires daily dosing, can cause side effects and does not eradicate the infection3,4. Although anti-HIV-1 antibodies constitute a potential alternative to ART5,6, treatment of viremic individuals with a single antibody also results in emergence of resistant viral variants7-9. Moreover, combinations of first-generation anti-HIV-1 broadly neutralizing antibodies (bNAbs) had little measurable effect on the infection10-12. Here we report on a phase 1b clinical trial ( NCT02825797 ) in which two potent bNAbs, 3BNC11713 and 10-107414, were administered in combination to seven HIV-1 viremic individuals. Infusions of 30 mg kg-1 of each of the antibodies were well-tolerated. In the four individuals with dual antibody-sensitive viruses, immunotherapy resulted in an average reduction in HIV-1 viral load of 2.05 log10 copies per ml that remained significantly reduced for three months following the first of up to three infusions. In addition, none of these individuals developed resistance to both antibodies. Larger studies will be necessary to confirm the efficacy of antibody combinations in reducing HIV-1 viremia and limiting the emergence of resistant viral variants.


Subject(s)
Antibodies, Neutralizing/administration & dosage , HIV Infections/drug therapy , Immunotherapy , Viremia/drug therapy , Adolescent , Adult , Aged , Antibodies, Neutralizing/adverse effects , Antiretroviral Therapy, Highly Active , Female , HIV Infections/virology , HIV Seropositivity , HIV-1/pathogenicity , Humans , Male , Middle Aged , Viral Load/drug effects , Viremia/virology , Young Adult
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