Correction: Histone Demethylase JMJD2B Functions as a Co-Factor of Estrogen Receptor in Breast Cancer Proliferation and Mammary Gland Development.

[This corrects the article DOI: 10.1371/journal.pone.0017830.].

Modification of the HLA-A*24:02 Peptide Binding Pocket Enhances Cognate Peptide-Binding Capacity and Antigen-Specific T Cell Activation.

The immunogenicity of a T cell Ag is correlated with the ability of its antigenic epitope to bind HLA and be stably presented to T cells. This presents a challenge for the development of effective cancer immunotherapies, as many self-derived tumor-associated epitopes elicit weak T cell responses, in part due to weak binding affinity to HLA. Traditional methods to increase peptide-HLA binding affinity involve modifying the peptide to reflect HLA allele binding preferences. Using a different approach, we sought to analyze whether the immunogenicity of wild-type peptides could be altered through modification of the HLA binding pocket. After analyzing HLA class I peptide binding pocket alignments, we identified an alanine 81 to leucine (A81L) modification within the F binding pocket of HLA-A*24:02 that was found to heighten the ability of artificial APCs to retain and present HLA-A*24:02-restricted peptides, resulting in increased T cell responses while retaining Ag specificity. This modification led to increased peptide exchange efficiencies for enhanced detection of low-avidity T cells and, when expressed on artificial APCs, resulted in greater expansion of Ag-specific T cells from melanoma-derived tumor-infiltrating lymphocytes. Our study provides an example of how modifications to the HLA binding pocket can enhance wild-type cognate peptide presentation to heighten T cell activation.

Affinity-matured HLA class II dimers for robust staining of antigen-specific CD4+ T cells.

Peptide-major histocompatibility complex (pMHC) multimers enable the detection of antigen-specific T cells in studies ranging from vaccine efficacy to cancer immunotherapy. However, this technology is unreliable when applied to pMHC class II for the detection of CD4+ T cells. Here, using a combination of molecular biological and immunological techniques, we cloned sequences encoding human leukocyte antigen (HLA)-DP, HLA-DQ and HLA-DR molecules with enhanced CD4 binding affinity (with a Kd of 8.9 ± 1.1 µM between CD4 and affinity-matured HLA-DP4) and produced affinity-matured class II dimers that stain antigen-specific T cells better than conventional multimers in both in vitro and ex vivo analyses. Using a comprehensive library of dimers for HLA-DP4, which is the most frequent HLA allele in many ancestry groups, we mapped 103 HLA-DP4-restricted epitopes derived from diverse tumor-associated antigens and cloned the cognate T-cell antigen receptor (TCR) genes from in vitro-stimulated CD4+ T cells. The availability of affinity-matured class II dimers across HLA-DP, HLA-DQ and HLA-DR alleles will aid in the investigation of human CD4+ T-cell responses.

Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy.

Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA, and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy.

Landscape mapping of shared antigenic epitopes and their cognate TCRs of tumor-infiltrating T lymphocytes in melanoma.

HLA-restricted T cell responses can induce antitumor effects in cancer patients. Previous human T cell research has largely focused on the few HLA alleles prevalent in a subset of ethnic groups. Here, using a panel of newly developed peptide-exchangeable peptide/HLA multimers and artificial antigen-presenting cells for 25 different class I alleles and greater than 800 peptides, we systematically and comprehensively mapped shared antigenic epitopes recognized by tumor-infiltrating T lymphocytes (TILs) from eight melanoma patients for all their class I alleles. We were able to determine the specificity, on average, of 12.2% of the TILs recognizing a mean of 3.1 shared antigen-derived epitopes across HLA-A, B, and C. Furthermore, we isolated a number of cognate T cell receptor genes with tumor reactivity. Our novel strategy allows for a more complete examination of the immune response and development of novel cancer immunotherapy not limited by HLA allele prevalence or tumor mutation burden.

The immune system is the body's way of defending itself, offering protection against diseases such as cancer. But to remove the cancer cells, the immune system must be able to identify them as different from the rest of the body. All cells break down proteins into shorter fragments, known as peptides, that are displayed on the cell surface by a protein called human leukocyte antigen, HLA for short. Cancer cells display distinctive peptides on their surface as they generate different proteins to those of healthy cells. Immune cells called T cells use these abnormal peptides to identify the cancer so that it can be destroyed. Sometimes T cells can lack the right equipment to detect abnormal peptides, allowing cancer cells to hide from the immune system. However, T cells can be trained through a treatment called immunotherapy, which provides T cells with new tools so that they can spot the peptides displayed by HLA on the previously 'hidden' cancer cells. There are many different forms of HLA, each of which can display different peptides. Current research in immunotherapy commonly targets only a subset of HLA forms, and not all cancer patients have these types. This means that immunotherapy research is only likely to be of most benefit to a limited number of patients. Immunotherapy could be made effective for more people if new cancer peptides that are displayed by the other 'under-represented' forms of HLA were identified. Murata, Nakatsugawa et al. have now used T cells that were taken from tumors in eight patients with melanoma, which is a type of skin cancer. A library of fluorescent HLA-peptides was generated ­ using a new, simplified methodology ­ with 25 forms of HLA that displayed over 800 peptides. T cells were then mixed with the library to identify which HLA-peptides they can target. As a result, Murata, Nakatsugawa et al. found the cancer targets of around 12% of the tumor-infiltrating T cells tested, including those from under-represented forms of HLA. Consequently, these findings could be used to develop new immunotherapies that can treat more patients.

Chaperones of the class I peptide-loading complex facilitate the constitutive presentation of endogenous antigens on HLA-DP84GGPM87.

Recent work has delineated key differences in the antigen processing and presentation mechanisms underlying HLA-DP alleles encoding glycine at position 84 of the DPß chain (DP84GGPM87). These DPs are unable to associate with the class II-associated Ii peptide (CLIP) region of the invariant chain (Ii) chaperone early in the endocytic pathway, leading to continuous presentation of endogenous antigens. However, little is known about the chaperone support involved in the loading of these endogenous antigens onto DP molecules. Here, we demonstrate the proteasome and TAP dependency of this pathway and reveal the ability of HLA class I to compete with DP84GGPM87 for the presentation of endogenous antigens, suggesting that shared subcellular machinery may exist between the two classes of HLA. We identify physical interactions of prototypical class I-associated chaperones with numerous DP alleles, including TAP2, tapasin, ERp57, calnexin, and calreticulin, using a conventional immunoprecipitation and immunoblot approach and confirm the existence of these interactions in vivo through the use of the BioID2 proximal biotinylation system in human cells. Based on immunological assays, we then demonstrate the ability of each of these chaperones to facilitate the presentation of endogenously derived, but not exogenously derived, antigens on DP molecules. Considering previous genetic and clinical studies linking DP84GGPM87 to disease frequency and severity in autoimmune disease, viral infections, and cancer, we suggest that the above chaperones may form the molecular basis of these observable clinical differences through facilitating the presentation of endogenously derived antigens to CD4+ T cells.

Arginine methylation of FOXP3 is crucial for the suppressive function of regulatory T cells.

Forkhead box transcription factor 3 (FOXP3) plays a pivotal role in the suppressive function of regulatory T cells. In addition to mRNA levels, FOXP3 activity can also be controlled by posttranslational mechanisms, which have not been studied in a comprehensive manner. Through extensive screening using selective inhibitors, we demonstrate that the inhibition of type I protein arginine methytransferases (PRMTs) attenuates the suppressive functions of regulatory T cells. FOXP3 undergoes methylation on arginine residues at positions 48 and 51 by interacting with protein arginine methyltransferase 1 (PRMT1). The inhibition of arginine methylation confers gene expression profiles representing type I helper T cells to FOXP3+ T cells, which results in attenuated suppressive activity. A methylation-defective mutant of FOXP3 displays less potent activity to suppress xenogeneic graft-versus-host disease in vivo. These results elucidate an important role of arginine methylation to enhance FOXP3 functions and are potentially applicable to modulate regulatory T cell functions.

JMJD2B/KDM4B inactivation in adipose tissues accelerates obesity and systemic metabolic abnormalities.

Obesity is a serious global health issue; however, the roles of genetics and epigenetics in the onset and progression of obesity are still not completely understood. The aim of this study was to determine the role of Kdm4b, which belongs to a subfamily of histone demethylases, in adipogenesis and fat metabolism in vivo. We established conditional Kdm4b knockout mice. Inactivation of Kdm4b in adipocytes (K4bKO) induced profound obesity in mice on a high fat diet (HFD). The HFD-fed K4bKO mice exhibited an increased volume of fat mass and higher expression levels of adipogenesis-related genes. In contrast, the genes involved in energy expenditure and mitochondrial functions were down-regulated. Supporting these findings, the energy expenditure of Kdm4b-deficient cells was markedly decreased. In addition, progression of glucose intolerance and hepatic steatosis with hepatocellular damages was observed. These data indicate that Kdm4b is a critical regulator of systemic metabolism via enhancing energy expenditure in adipocytes.

DOT1L inhibition attenuates graft-versus-host disease by allogeneic T cells in adoptive immunotherapy models.

Adoptive T-cell therapy is a promising therapeutic approach for cancer patients. The use of allogeneic T-cell grafts will improve its applicability and versatility provided that inherent allogeneic responses are controlled. T-cell activation is finely regulated by multiple signaling molecules that are transcriptionally controlled by epigenetic mechanisms. Here we report that inhibiting DOT1L, a histone H3-lysine 79 methyltransferase, alleviates allogeneic T-cell responses. DOT1L inhibition reduces miR-181a expression, which in turn increases the ERK phosphatase DUSP6 expression and selectively ameliorates low-avidity T-cell responses through globally suppressing T-cell activation-induced gene expression alterations. The inhibition of DOT1L or DUSP6 overexpression in T cells attenuates the development of graft-versus-host disease, while retaining potent antitumor activity in xenogeneic and allogeneic adoptive immunotherapy models. These results suggest that DOT1L inhibition may enable the safe and effective use of allogeneic antitumor T cells by suppressing unwanted immunological reactions in adoptive immunotherapy.

Mechanisms underlying the lack of endogenous processing and CLIP-mediated binding of the invariant chain by HLA-DP84Gly.

While the principles of classical antigen presentation via MHC class II are well-established, the mechanisms for the many routes of cross-presentation by which endogenous antigens become associated with class II molecules are not fully understood. We have recently demonstrated that the single amino acid polymorphism HLA-DPß84Gly (DP84Gly) is critical to abrogate class II invariant chain associated peptide (CLIP) region-mediated binding of invariant chain (Ii) to DP, allowing endoplasmic reticulum (ER)-resident endogenous antigens to constitutively associate with DP84Gly such as DP4. In this study, we demonstrate that both the CLIP and N-terminal non-CLIP Ii regions cooperatively generate an Ii conformation that cannot associate with DP84Gly via the CLIP region. We also demonstrate the ability of DP4 to efficiently process and present antigens encoded in place of CLIP in a chimeric Ii, regardless of wild type Ii and HLA-DM expression. These data highlight the complex interplay between DP polymorphisms and the multiple Ii regions that cooperatively regulate this association, ultimately controlling the presentation of endogenous antigens on DP molecules. These results may also offer a mechanistic explanation for recent studies identifying the differential effects between DP84Gly and DP84Asp as clinically relevant in human disease.

A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects.

The adoptive transfer of T cells engineered with a chimeric antigen receptor (CAR) (hereafter referred to as CAR-T cells) specific for the B lymphocyte antigen CD19 has shown impressive clinical responses in patients with refractory B cell malignancies. However, the therapeutic effects of CAR-T cells that target other malignancies have not yet resulted in significant clinical benefit. Although inefficient tumor trafficking and various immunosuppressive mechanisms can impede CAR-T cell effector responses, the signals delivered by the current CAR constructs may still be insufficient to fully activate antitumor T cell functions. Optimal T cell activation and proliferation requires multiple signals, including T cell receptor (TCR) engagement (signal 1), co-stimulation (signal 2) and cytokine engagement (signal 3). However, CAR constructs currently being tested in the clinic contain a CD3z (TCR signaling) domain and co-stimulatory domain(s) but not a domain that transmits signal 3 (refs. 13, 14, 15, 16, 17, 18). Here we have developed a novel CAR construct capable of inducing cytokine signaling after antigen stimulation. This new-generation CD19 CAR encodes a truncated cytoplasmic domain from the interleukin (IL)-2 receptor ß-chain (IL-2Rß) and a STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif, together with the TCR signaling (CD3z) and co-stimulatory (CD28) domains (hereafter referred to as 28-ΔIL2RB-z(YXXQ)). The 28-ΔIL2RB-z(YXXQ) CAR-T cells showed antigen-dependent activation of the JAK kinase and of the STAT3 and STAT5 transcription factors signaling pathways, which promoted their proliferation and prevented terminal differentiation in vitro. The 28-ΔIL2RB-z(YXXQ) CAR-T cells demonstrated superior in vivo persistence and antitumor effects in models of liquid and solid tumors as compared with CAR-T cells expressing a CD28 or 4-1BB co-stimulatory domain alone. Taken together, these results suggest that our new-generation CAR has the potential to demonstrate superior antitumor effects with minimal toxicity in the clinic and that clinical translation of this novel CAR is warranted.

A Subset of Human Autoreactive CD1c-Restricted T Cells Preferentially Expresses TRBV4-1+ TCRs.

In humans, a substantial portion of T cells recognize lipids presented by the monomorphic CD1 proteins. Recent studies have revealed the molecular basis of mycobacterial lipid recognition by CD1c-restricted T cells. Subsets of CD1c-restricted T cells recognize self-lipids in addition to foreign lipids, which may have implications in human diseases involving autoimmunity and malignancy. However, the molecular identity of these self-reactive T cells remains largely elusive. In this study, using a novel CD1c+ artificial APC (aAPC)-based system, we isolated human CD1c-restricted autoreactive T cells and characterized them at the molecular level. By using the human cell line K562, which is deficient in MHC class I/II and CD1 expression, we generated an aAPC expressing CD1c as the sole Ag-presenting molecule. When stimulated with this CD1c+ aAPC presenting endogenous lipids, a subpopulation of primary CD4+ T cells from multiple donors was consistently activated, as measured by CD154 upregulation and cytokine production in a CD1c-specific manner. These activated CD4+ T cells preferentially expressed TRBV4-1+ TCRs. Clonotypic analyses of the reconstituted TRBV4-1+ TCR genes confirmed CD1c-restricted autoreactivity of this repertoire, and the strength of CD1c reactivity was influenced by the diversity of CDR3ß sequences. Finally, alanine scanning of CDR1 and CDR2 sequences of TRBV4-1 revealed two unique residues, Arg30 and Tyr51, as critical in conferring CD1c-restricted autoreactivity, thus elucidating the molecular basis of the observed V gene bias. These data provide new insights into the molecular identity of human autoreactive CD1c-restricted T cells.

HLA-DP84Gly constitutively presents endogenous peptides generated by the class I antigen processing pathway.

Classical antigen processing leads to the presentation of antigenic peptides derived from endogenous and exogenous sources for MHC class I and class II molecules, respectively. Here we show that, unlike other class II molecules, prevalent HLA-DP molecules with ß-chains encoding Gly84 (DP84Gly) constitutively present endogenous peptides. DP84Gly does not bind invariant chain (Ii) via the class II-associated invariant chain peptide (CLIP) region, nor does it present CLIP. However, Ii does facilitate the transport of DP84Gly from the endoplasmic reticulum (ER) to the endosomal/lysosomal pathway by transiently binding DP84Gly via a non-CLIP region(s) in a pH-sensitive manner. Accordingly, like class I, DP84Gly constitutively presents endogenous peptides processed by the proteasome and transported to the ER by the transporter associated with antigen processing (TAP). Therefore, DP84Gly, found only in common chimpanzees and humans, uniquely uses both class I and II antigen-processing pathways to present peptides derived from intracellular and extracellular sources.

Transient stimulation expands superior antitumor T cells for adoptive therapy.

Adoptive cell therapy is a potentially curative therapeutic approach for patients with cancer. In this treatment modality, antitumor T cells are exponentially expanded in vitro prior to infusion. Importantly, the results of recent clinical trials suggest that the quality of expanded T cells critically affects their therapeutic efficacy. Although anti-CD3 mAb-based stimulation is widely used to expand T cells in vitro, a protocol to generate T cell grafts for optimal adoptive therapy has yet to be established. In this study, we investigated the differences between T cell stimulation mediated by anti-CD3/CD28 mAb-coated beads and cell-based artificial antigen-presenting cells (aAPCs) expressing CD3/CD28 counter-receptors. We found that transient stimulation with cell-based aAPCs, but not prolonged stimulation with beads, resulted in the superior expansion of CD8+ T cells. Transiently stimulated CD8+ T cells maintained a stem cell-like memory phenotype and were capable of secreting multiple cytokines significantly more efficiently than chronically stimulated T cells. Importantly, the chimeric antigen receptor-engineered antitumor CD8+ T cells expanded via transient stimulation demonstrated superior persistence and antitumor responses in adoptive immunotherapy mouse models. These results suggest that restrained stimulation is critical for generating T cell grafts for optimal adoptive immunotherapy for cancer.

Key Residues at Third CDR3ß Position Impact Structure and Antigen Recognition of Human Invariant NK TCRs.

The human invariant NK (iNK) TCR is largely composed of the invariant TCR Vα24-Jα18 chain and semivariant TCR Vß11 chains with variable CDR3ß sequences. The direct role of CDR3ß in Ag recognition has been studied extensively. Although it was noted that CDR3ß can interact with CDR3α, how this interaction might indirectly influence Ag recognition is not fully elucidated. We observed that the third position of Vß11 CDR3 can encode an Arg or Ser residue as a result of somatic rearrangement. Clonotypic analysis of the two iNK TCR types with a single amino acid substitution revealed that the staining intensity by anti-Vα24 Abs depends on whether Ser or Arg is encoded. When stained with an anti-Vα24-Jα18 Ab, human primary invariant NKT cells could be divided into Vα24 low- and high-intensity subsets, and Arg-encoding TCR Vß11 chains were more frequently isolated from the Vα24 low-intensity subpopulation compared with the Vα24 high-intensity subpopulation. The Arg/Ser substitution also influenced Ag recognition as determined by CD1d multimer staining and CD1d-restricted functional responses. Importantly, in silico modeling validated that this Ser-to-Arg mutation could alter the structure of the CDR3ß loop, as well as the CDR3α loop. Collectively, these results indicate that the Arg/Ser encoded at the third CDR3ß residue can effectively modulate the overall structure of, and Ag recognition by, human iNK TCRs.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain.

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCRα or TCRß hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

BET bromodomain inhibition enhances T cell persistence and function in adoptive immunotherapy models.

Adoptive immunotherapy is a potentially curative therapeutic approach for patients with advanced cancer. However, the in vitro expansion of antitumor T cells prior to infusion inevitably incurs differentiation towards effector T cells and impairs persistence following adoptive transfer. Epigenetic profiles regulate gene expression of key transcription factors over the course of immune cell differentiation, proliferation, and function. Using comprehensive screening of chemical probes with defined epigenetic targets, we found that JQ1, an inhibitor of bromodomain and extra-terminal motif (BET) proteins, maintained CD8+ T cells with functional properties of stem cell-like and central memory T cells. Mechanistically, the BET protein BRD4 directly regulated expression of the transcription factor BATF in CD8+ T cells, which was associated with differentiation of T cells into an effector memory phenotype. JQ1-treated T cells showed enhanced persistence and antitumor effects in murine T cell receptor and chimeric antigen receptor gene therapy models. Furthermore, we found that histone acetyltransferase p300 supported the recruitment of BRD4 to the BATF promoter region, and p300 inhibition similarly augmented antitumor effects of the adoptively transferred T cells. These results demonstrate that targeting the BRD4-p300 signaling cascade supports the generation of superior antitumor T cell grafts for adoptive immunotherapy.

CD4(+) and CD8(+) TCRß repertoires possess different potentials to generate extraordinarily high-avidity T cells.

Recent high throughput sequencing analysis has revealed that the TCRß repertoire is largely different between CD8(+) and CD4(+) T cells. Here, we show that the transduction of SIG35α, the public chain-centric HLA-A*02:01(A2)/MART127-35 TCRα hemichain, conferred A2/MART127-35 reactivity to a substantial subset of both CD8(+) and CD4(+) T cells regardless of their HLA-A2 positivity. T cells individually reconstituted with SIG35α and different A2/MART127-35 TCRß genes isolated from CD4(+) or CD8(+) T cells exhibited a wide range of avidity. Surprisingly, approximately half of the A2/MART127-35 TCRs derived from CD4(+) T cells, but none from CD8(+) T cells, were stained by A2/MART127-35 monomer and possessed broader cross-reactivity. Our results suggest that the differences in the primary structure of peripheral CD4(+) and CD8(+) TCRß repertoire indeed result in the differences in their ability to form extraordinarily high avidity T cells which would otherwise have been deleted by central tolerance.

The Histone Variant MacroH2A1.2 Is Necessary for the Activation of Muscle Enhancers and Recruitment of the Transcription Factor Pbx1.

Histone variants complement and integrate histone post-translational modifications in regulating transcription. The histone variant macroH2A1 (mH2A1) is almost three times the size of its canonical H2A counterpart, due to the presence of an ∼25 kDa evolutionarily conserved non-histone macro domain. Strikingly, mH2A1 can mediate both gene repression and activation. However, the molecular determinants conferring these alternative functions remain elusive. Here, we report that mH2A1.2 is required for the activation of the myogenic gene regulatory network and muscle cell differentiation. H3K27 acetylation at prospective enhancers is exquisitely sensitive to mH2A1.2, indicating a role of mH2A1.2 in imparting enhancer activation. Both H3K27 acetylation and recruitment of the transcription factor Pbx1 at prospective enhancers are regulated by mH2A1.2. Overall, our findings indicate a role of mH2A1.2 in marking regulatory regions for activation.