Pleun Hombrink

Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers.

The use of fluorescently labeled major histocompatibility complex multimers has become an essential technique for analyzing disease- and therapy-induced T-cell immunity. Whereas classical major histocompatibility complex multimer analyses are well-suited for the detection of immune responses to a few epitopes, limitations on human-subject sample size preclude a comprehensive analysis of T-cell immunity. To address this issue, we developed a combinatorial encoding strategy that allows the parallel detection of a multitude of different T-cell populations in a single sample. Detection of T cells from peripheral blood by combinatorial encoding is as efficient as detection with conventionally labeled multimers but results in a substantially increased sensitivity and, most notably, allows comprehensive screens to be performed. We obtained proof of principle for the feasibility of large-scale screening of human material by analysis of human leukocyte antigen A3–restricted T-cell responses to known and potential melanoma-associated antigens in peripheral blood from individuals with melanoma.

IL-1β, IL-4 and IL-12 control the fate of group 2 innate lymphoid cells in human airway inflammation in the lungs

Group 2 innate lymphoid cells (ILC2s) secrete type 2 cytokines, which protect against parasites but can also contribute to a variety of inflammatory airway diseases. We report here that interleukin 1β (IL-1β) directly activated human ILC2s and that IL-12 induced the conversion of these activated ILC2s into interferon-γ (IFN-γ)-producing ILC1s, which was reversed by IL-4. The plasticity of ILCs was manifested in diseased tissues of patients with severe chronic obstructive pulmonary disease (COPD) or chronic rhinosinusitis with nasal polyps (CRSwNP), which displayed IL-12 or IL-4 signatures and the accumulation of ILC1s or ILC2s, respectively. Eosinophils were a major cellular source of IL-4, which revealed cross-talk between IL-5-producing ILC2s and IL-4-producing eosinophils. We propose that IL-12 and IL-4 govern ILC2 functional identity and that their imbalance results in the perpetuation of type 1 or type 2 inflammation.

High-throughput identification of antigen-specific TCRs by TCR gene capture

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen–reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

The Human Leukocyte Antigen–presented Ligandome of B Lymphocytes

Peptides presented by human leukocyte antigen (HLA) molecules on the cell surface play a crucial role in adaptive immunology, mediating the communication between T cells and antigen presenting cells. Knowledge of these peptides is of pivotal importance in fundamental studies of T cell action and in cellular immunotherapy and transplantation. In this paper we present the in-depth identification and relative quantification of 14,500 peptide ligands constituting the HLA ligandome of B cells. This large number of identified ligands provides general insight into the presented peptide repertoire and antigen presentation. Our uniquely large set of HLA ligands allowed us to characterize in detail the peptides constituting the ligandome in terms of relative abundance, peptide length distribution, physicochemical properties, binding affinity to the HLA molecule, and presence of post-translational modifications. The presented B-lymphocyte ligandome is shown to be a rich source of information by the presence of minor histocompatibility antigens, virus-derived epitopes, and post-translationally modified HLA ligands, and it can be a good starting point for solving a wealth of specific immunological questions. These HLA ligands can form the basis for reversed immunology approaches to identify T cell epitopes based not on in silico predictions but on the bona fide eluted HLA ligandome.

Programs for the persistence, vigilance and control of human CD8+ lung-resident memory T cells

Tissue-resident memory T cells (TRM cells) in the airways mediate protection against respiratory infection. We characterized TRM cells expressing integrin αE (CD103) that reside within the epithelial barrier of human lungs. These cells had specialized profiles of chemokine receptors and adhesion molecules, consistent with their unique localization. Lung TRM cells were poised for rapid responsiveness by constitutive expression of deployment-ready mRNA encoding effector molecules, but they also expressed many inhibitory regulators, suggestive of programmed restraint. A distinct set of transcription factors was active in CD103+ TRM cells, including Notch. Genetic and pharmacological experiments with mice revealed that Notch activity was required for the maintenance of CD103+ TRM cells. We have thus identified specialized programs underlying the residence, persistence, vigilance and tight control of human lung TRM cells.

High-throughput identification of potential minor histocompatibility antigens by MHC tetramer-based screening: feasibility and limitations.

T-cell recognition of minor histocompatibility antigens (MiHA) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, the number of MiHA identified to date remains limited, making clinical application of MiHA reactive T-cell infusion difficult. This study represents the first attempt of genome-wide prediction of MiHA, coupled to the isolation of T-cell populations that react with these antigens. In this unbiased high-throughput MiHA screen, both the possibilities and pitfalls of this approach were investigated. First, 973 polymorphic peptides expressed by hematopoietic stem cells were predicted and screened for HLA-A2 binding. Subsequently a set of 333 high affinity HLA-A2 ligands was identified and post transplantation samples from allo-SCT patients were screened for T-cell reactivity by a combination of pMHC-tetramer-based enrichment and multi-color flow cytometry. Using this approach, 71 peptide-reactive T-cell populations were generated. The isolation of a T-cell line specifically recognizing target cells expressing the MAP4K1IMA antigen demonstrates that identification of MiHA through this approach is in principle feasible. However, with the exception of the known MiHA HMHA1, none of the other T-cell populations that were generated demonstrated recognition of endogenously MiHA expressing target cells, even though recognition of peptide-loaded targets was often apparent. Collectively these results demonstrate the technical feasibility of high-throughput analysis of antigen-specific T-cell responses in small patient samples. However, the high-sensitivity of this approach requires the use of potential epitope sets that are not solely based on MHC binding, to prevent the frequent detection of T-cell responses that lack biological relevance.

Allogeneic HLA-A*02–Restricted WT1-Specific T Cells from Mismatched Donors Are Highly Reactive but Show Off-Target Promiscuity

T cells recognizing tumor-associated Ags such as Wilms tumor protein (WT1) are thought to exert potent antitumor reactivity. However, no consistent high-avidity T cell responses have been demonstrated in vaccination studies with WT1 as target in cancer immunotherapy. The aim of this study was to investigate the possible role of negative thymic selection on the avidity and specificity of T cells directed against self-antigens. T cell clones directed against the HLA-A*0201–binding WT1126–134 peptide were generated from both HLA-A*02–positive (self-HLA–restricted) and HLA-A*02–negative [nonself (allogeneic) HLA [allo-HLA]-restricted] individuals by direct ex vivo isolation using tetramers or after in vitro priming and selection. The functional avidity and specificity of these T cell clones was analyzed in-depth. Self-HLA–restricted WT1-specific clones only recognized WT1126–134 with low avidities. In contrast, allo-HLA–restricted WT1 clones exhibited profound functional reactivity against a multitude of HLA-A*02–positive targets, even in the absence of exogenously loaded WT1 peptide, indicative of Ag-binding promiscuity. To characterize this potential promiscuity, reactivity of the T cell clones against 400 randomly selected HLA-A*0201–binding peptides was investigated. The self-HLA–restricted WT1-specific T cell clones only recognized the WT1 peptide. In contrast, the allo-HLA–restricted WT1-reactive clones recognized besides WT1 various other HLA-A*0201–binding peptides. In conclusion, allogeneic HLA-A*02–restricted WT1-specific T cells isolated from mismatched donors may be more tumor-reactive than their autologous counterparts but can show specific off-target promiscuity of potential clinical importance. As a result of this, administration of WT1-specific T cells generated from HLA-mismatched donors should be performed with appropriate precautions against potential off-target effects.

Discovery of T Cell Epitopes Implementing HLA-Peptidomics into a Reverse Immunology Approach

T cell recognition of minor histocompatibility Ags (MiHA) plays an important role in the graft-versus-tumor effect of allogeneic stem cell transplantation. Selective infusion of T cells reactive for hematopoiesis-restricted MiHA presented in the context of HLA class I or II molecules may help to separate the graft-versus-tumor effects from graft-versus-host disease effects after allogeneic stem cell transplantation. Over the years, increasing numbers of MiHA have been identified by forward immunology approaches, and the relevance of these MiHA has been illustrated by correlation with clinical outcome. As the tissue distribution of MiHA affects the clinical outcome of T cell responses against these Ags, it would be beneficial to identify additional predefined MiHA that are exclusively expressed on hematopoietic cells. Therefore, several reverse immunology approaches have been explored for the prediction of MiHA. Thus far, these approaches frequently resulted in the identification of T cells directed against epitopes that are not naturally processed and presented. In this study we established a method for the identification of biologically relevant MiHA, implementing mass spectrometry–based HLA-peptidomics into a reverse immunology approach. For this purpose, HLA class I binding peptides were eluted from transformed B cells, analyzed by mass spectrometry, and matched with a database dedicated to identifying polymorphic peptides. This process resulted in a set of 40 MiHA candidates that were evaluated in multiple selection steps. The identification of LB-NISCH-1A demonstrated the technical feasibility of our approach. On the basis of these results, we present an approach that can be of value for the efficient identification of MiHA or other T cell epitopes.

Mixed functional characteristics correlating with TCR-ligand koff -rate of MHC-tetramer reactive T cells within the naive T-cell repertoire.

The low frequency of antigen‐specific naïve T cells has challenged numerous laboratories to develop various techniques to study the naïve T‐cell repertoire. Here, we combine the generation of naïve repertoire‐derived antigen‐specific T‐cell lines based on MHC‐tetramer staining and magnetic‐bead enrichment with in‐depth functional assessment of the isolated T cells. Cytomegalovirus (CMV) specific T‐cell lines were generated from seronegative individuals. Generated T‐cell lines consisted of a variety of immunodominant CMV‐epitope‐specific oligoclonal T‐cell populations restricted to various HLA‐molecules (HLA‐A1, A2, B7, B8, and B40), and the functional and structural avidity of the CMV‐specific T cells was studied. Although all CMV‐specific T cells were isolated based on their reactivity toward a specific peptide‐MHC complex, we observed a large variation in the functional avidity of the MHC‐tetramer positive T‐cell populations, which correlated with the structural avidity measured by the recently developed Streptamer koff‐rate assay. Our data demonstrate that MHC‐tetramer staining is not always predictive for specific T‐cell reactivity, and challenge the sole use of MHC‐tetramers as an indication of the peripheral T‐cell repertoire, independent of the analysis of functional activity or structural avidity parameters.

The Transcription Factor Hobit Identifies Human Cytotoxic CD4(+) T Cells

The T cell lineage is commonly divided into CD4-expressing helper T cells that polarize immune responses through cytokine secretion and CD8-expressing cytotoxic T cells that eliminate infected target cells by virtue of the release of cytotoxic molecules. Recently, a population of CD4+ T cells that conforms to the phenotype of cytotoxic CD8+ T cells has received increased recognition. These cytotoxic CD4+ T cells display constitutive expression of granzyme B and perforin at the protein level and mediate HLA class II-dependent killing of target cells. In humans, this cytotoxic profile is found within the human cytomegalovirus (hCMV)-specific, but not within the influenza- or Epstein–Barr virus-specific CD4+ T cell populations, suggesting that, in particular, hCMV infection induces the formation of cytotoxic CD4+ T cells. We have previously described that the transcription factor Homolog of Blimp-1 in T cells (Hobit) is specifically upregulated in CD45RA+ effector CD8+ T cells that arise after hCMV infection. Here, we describe the expression pattern of Hobit in human CD4+ T cells. We found Hobit expression in cytotoxic CD4+ T cells and accumulation of Hobit+ CD4+ T cells after primary hCMV infection. The Hobit+ CD4+ T cells displayed highly overlapping characteristics with Hobit+ CD8+ T cells, including the expression of cytotoxic molecules, T-bet, and CX3CR1. Interestingly, γδ+ T cells that arise after hCMV infection also upregulate Hobit expression and display a similar effector phenotype as cytotoxic CD4+ and CD8+ T cells. These findings suggest a shared differentiation pathway in CD4+, CD8+, and γδ+ T cells that may involve Hobit-driven acquisition of long-lived cytotoxic effector function.