Search

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses1–3; however, the relationship between phenotypic characteristics and TCR properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8+ T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide–human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire. The authors use single-cell profiling and T cell receptor specificity screening to show how tumour antigen recognition shapes the phenotypes of CD8+ T cells and antitumour immune responses.

Within the tumour microenvironment, CD4(+) T cells can promote or suppress antitumour responses through the recognition of antigens presented by human leukocyte antigen (HLA) class II molecules(1,2), but how cancers co-opt these physiologic processes to achieve immune evasion remains incompletely understood. Here we performed in-depth analysis of the phenotype and tumour specificity of CD4(+) T cells infiltrating human melanoma specimens, finding that exhausted cytotoxic CD4(+) T cells could be directly induced by melanoma cells through recognition of HLA class II-restricted neoantigens, and also HLA class I-restricted tumour-associated antigens. CD4(+) T regulatory (T(Reg)) cells could be indirectly elicited through presentation of tumour antigens via antigen-presenting cells. Notably, numerous tumour-reactive CD4(+) T(Reg) clones were stimulated directly by HLA class II-positive melanoma and demonstrated specificity for melanoma neoantigens. This phenomenon was observed in the presence of an extremely high tumour neoantigen load, which we confirmed to be associated with HLA class II positivity through the analysis of 116 melanoma specimens. Our data reveal the landscape of infiltrating CD4(+) T cells in melanoma and point to the presentation of HLA class II-restricted neoantigens and direct engagement of immunosuppressive CD4(+) T(Reg) cells as a mechanism of immune evasion that is favoured in HLA class II-positive melanoma.

Cancer vaccines aim to direct the immune system to eradicate cancer cells. Here we review the essential immunologic concepts underpinning natural immunity and highlight the multiple unique challenges faced by vaccines targeting cancer. Recent technological advances in mass spectrometry, neoantigen prediction, genetically and pharmacologically engineered mouse models, and single-cell omics have revealed new biology, which can help to bridge this divide. We particularly focus on translationally relevant aspects, such as antigen selection and delivery and the monitoring of human post-vaccination responses, and encourage more aggressive exploration of novel approaches.

Targeting immune checkpoint pathways, such as programmed death ligand-1 (PD-L1, also known as CD274 or B7-H1) or its receptor programmed cell death-1 (PD-1) has shown improved survival for patients with numerous types of cancers, not limited to lung cancer, melanoma, renal cell carcinoma, and Hodgkin lymphoma. PD-L1 is a co-inhibitory molecule whose expression on the surface of tumor cells is associated with worse prognosis in many tumors. Here we describe a splice variant (secPD-L1) that does not splice into the transmembrane domain, but instead produces a secreted form of PD-L1 that has a unique 18 amino acid tail containing a cysteine that allows it to homodimerize and more effectively inhibit lymphocyte function than monomeric soluble PD-L1. We show that recombinant secPD-L1 can dimerize and inhibit T-cell proliferation and IFN-gamma production in vitro. The secPD-L1 variant is expressed by malignant cells in vitro that also express high levels of full-length PD-L1. Transcriptomic analysis of gene expression across The Cancer Genome Atlas found the strongest association of secPD-L1 with full-length PD-L1, but also with subsets of immunologic genes, such as in myeloid-derived suppressor cells. Moreover, the splice variant is also expressed in normal tissues and within normal peripheral blood cells it is preferentially expressed in activated myeloid cells. This is the first report of a form of secreted PD-L1 that homodimerizes and is functionally active. SecPD-L1 may function as a paracrine negative immune regulator within the tumor, since secPD-L1 does not require a cell-to-cell interaction to mediate its inhibitory effect. Electronic supplementary material The online version of this article (10.1007/s00262-018-2282-1) contains supplementary material, which is available to authorized users.

Personal neoantigen vaccines have been envisioned as an effective approach to induce, amplify and diversify antitumor T cell responses. To define the long-term effects of such a vaccine, we evaluated the clinical outcome and circulating immune responses of eight patients with surgically resected stage IIIB/C or IVM1a/b melanoma, at a median of almost 4 years after treatment with NeoVax, a long-peptide vaccine targeting up to 20 personal neoantigens per patient ( NCT01970358 ). All patients were alive and six were without evidence of active disease. We observed long-term persistence of neoantigen-specific T cell responses following vaccination, with ex vivo detection of neoantigen-specific T cells exhibiting a memory phenotype. We also found diversification of neoantigen-specific T cell clones over time, with emergence of multiple T cell receptor clonotypes exhibiting distinct functional avidities. Furthermore, we detected evidence of tumor infiltration by neoantigen-specific T cell clones after vaccination and epitope spreading, suggesting on-target vaccine-induced tumor cell killing. Personal neoantigen peptide vaccines thus induce T cell responses that persist over years and broaden the spectrum of tumor-specific cytotoxicity in patients with melanoma. Personalized neoantigen vaccination in patients with melanoma elicits durable and specific memory T cell clones that have cytotoxic gene signatures and can diversify to include nonvaccine neoantigen specificities.

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses

Summary: Cancer vaccine development has been historically fraught with difficulty, but tremendous progress has been made over the past 5 years. In this In Focus article, we reflect on the progress and challenges with vaccine development for cancers in general and for hematologic malignancies in particular, and suggest how our cancer vaccine experience can offer insight into COVID-19 vaccination.

Personal neoantigen–based cancer vaccines are designed to target antigens arising from tumor-specific mutations within individual cancers and present a tremendous opportunity to capitalize on their favorable and intrinsic properties of escape from central tolerance and exquisite tumor specificity. With the endpoint of creating an optimal T-cell army to attack a tumor, neoantigen-based vaccines have demonstrated the ability to coax naïve T-cell recruits against epitopes that do not induce spontaneous immunity to raise long-lasting T-cell responses against multiple tumor-specific epitopes and subsequently to extend the breadth of responses, as immunity begets immunity via epitope spreading. Importantly, on both preclinical and clinical fronts, the association of T-cell responses to neoantigens and favorable outcomes has been demonstrated time and time again. We recognize, however, that the path forward remains long and winding and requires the field to address several key challenges, particularly overcoming evolved tumor escape mechanisms and optimizing vaccine-induced immunity. Some challenges stem from gaps in science that enable in silico prediction of antigen presentation and recognition by T-cell receptors, whereas others stem from the logistical obstacles and cost of personalization. Nevertheless, with perseverance and innovative solutions, we have little doubt that the ability of neoantigen vaccination to induce potent cancer-specific T cells will fundamentally succeed in enabling greater effectiveness of a broad array of immunotherapies. We provide our perspective on the progress and the remaining challenges to realizing the opportunity of personal neoantigen cancer vaccines.

BackgroundWithin the tumor microenvironment, distinct CD4+ T cell subsets can play different and even opposite roles either promoting or suppressing anti-tumor responses through the recognition of antigens presented by human leukocyte antigen (HLA) class II molecules. However, how cancers co-opt these processes to shape the intratumoral CD4+ landscape and achieve immune evasion remains incompletely understood.MethodsWe performed single-cell characterization of CD4+ tumor infiltrating lymphocytes (TILs) collected from four human melanoma with low or high HLA-class II expression and we utilized TCR reconstruction and antigen specificity screening to unambiguously discover the tumor reactivity of CD4+ TILs. By testing TCR-transduced T cells against autologous patient-derived melanoma cell lines or against autologous antigen presenting cells (APCs) loaded with tumor lysates, we assessed the capacity of CD4+ TCRs to directly or indirectly recognize tumor cells. We defined the antigen-specificity of antitumor CD4+ TCRs by assessing their reactivity towards personal neoantigens (NeoAg) or public melanoma associated antigens (MAAs). Finally, we correlated NeoAg burden and HLA-class II expression in a series of 116 melanoma specimens from 4 independent cohorts of patients.ResultsAnalysis of single-cell data showed that the cluster distribution of cells within each CD4+ TCR clonotype family was highly homogeneous and appeared to follow 3 distinct major phenotypes, corresponding to non-exhausted memory cells, exhausted cells and regulatory cells (TRegs). Strikingly, clonally expanded CD4+ TReg-TILs were highly abundant within the tumor microenvironment of HLA class IIpos melanomas. We found that TCRs from exhausted cytotoxic CD4+ T cells could be directly triggered by melanoma cells not only through recognition of HLA class II restricted antigens, but also through presentation of HLA class I restricted MAAs. TReg-TCRs could be indirectly elicited through presentation of tumor antigens via APCs. Notably, numerous tumor-reactive CD4+ TReg-TCRs were directly stimulated by HLA class IIpos melanoma and demonstrated specificity for melanoma NeoAgs. In HLA class IIpos melanomas, the clonal expansion of numerous tumor-reactive and NeoAg-specific TRegs-clones appeared to be favored by a dramatically high tumor NeoAg load. Analysis of 116 melanoma specimens confirmed the association of elevated HLA-class II expression with extremely high NeoAg burden.ConclusionsOur data elucidate the landscape of infiltrating CD4+ T cells in melanoma and point to presentation of HLA-class II restricted NeoAgs and direct engagement of immunosuppressive CD4+ TRegs as a novel mechanism of immune evasion favored in HLA class IIpos melanoma.

Summary Increasing evidence indicates CD4+ T cells can recognize cancer-specific antigens and control tumor growth. However, it remains difficult to predict the antigens that will be presented by human leukocyte antigen class II molecules (HLA-II), hindering efforts to optimally target them therapeutically. Obstacles include inaccurate peptide-binding prediction and unsolved complexities of the HLA-II pathway. To address these challenges, we developed an improved technology for discovering HLA-II binding motifs and conducted a comprehensive analysis of tumor ligandomes to learn processing rules relevant in the tumor microenvironment. We profiled >40 HLA-II alleles and showed that binding motifs were highly sensitive to HLA-DM, a peptide-loading chaperone. We also revealed that intratumoral HLA-II presentation was dominated by professional antigen-presenting cells (APCs) rather than cancer cells. Integrating these observations, we developed algorithms that accurately predicted APC ligandomes, including peptides from phagocytosed cancer cells. These tools and biological insights will enable improved HLA-II-directed cancer therapies.

While tumor-reactive CD4+ T cells have been associated with effective immunotherapy responses, accurate prediction of MHC class II-restricted ligands remains a challenge, limiting our ability to harness CD4+ immunity for cancer therapy. To this end, we have developed a state-of-the-art MHC class II binding predictor, neonmhc2, trained on HLA-II ligands identified from mass spectrometry (MS) of cell lines engineered to express a single affinity-tagged HLA-II allele. Over 60 HLA-II alleles have been characterized to date. We demonstrate that neonmhc2 outperforms NetMHCIIpan, the current benchmark for class II prediction, in distinguishing 1) HLA-II ligands presented in cell lines and tissues, and critically, 2) immunogenic CD4+ epitopes identified with tetramer-guided epitope mapping. Furthermore, we show that numerous neoantigen peptides that were ranked highly by neonmhc2 but poorly by NetMHCIIpan were immunogenic in an ex vivo induction. We next studied HLA-II antigen processing in order to further boost our ability to predict CD4+ epitopes. We determined a gene-level bias by comparing transcript expression and gene length to the frequency of observations in MS, finding that secreted genes are over-represented in HLA-II ligandomes from tissues. We built numerous primary sequence-based processability predictors trained on MS data, but only achieved significant prediction improvement when using the simple feature of determining if a candidate peptide contained sequence that overlapped a previously observed HLA-II ligand. By combining neonmhc2 binding prediction, transcript expression, gene bias, and the overlap feature in an integrated presentation predictor, we were able to achieve up to a 61-fold increase in ability to predict HLA-II peptides presented from tissue MS data over NetMHCIIpan alone. We also sought to understand which cells are presenting HLA-II ligands in the tumor microenvironment to elucidate the presentation pathway most relevant to immunotherapy. By leveraging publicly available RNA-seq (bulk and single cell), we found that professional antigen-presenting cells rather than tumor cells are primarily responsible for HLA-II presentation. We developed a novel SILAC-based MS workflow to directly interrogate peptides derived from phagocytosed tumor cells that are presented by dendritic cells. The experiment revealed that mitochondrial genes are preferentially presented from phagocytosed cells. In conclusion, by integrating proteomics and genomics data at large scale, we have defined new rules for understanding HLA-II processing and presentation, particularly in the context of the tumor microenvironment. This work should enhance our ability to predict CD4+ epitopes for immunotherapy. Citation Format: Dewi Harjanto, Jennifer G. Abelin, Matthew Malloy, Prerna Suri, Tyler Colson, Scott P. Goulding, Amanda L. Creech, Lia R. Serrano, Gibbs Nasir, Yusuf Nasrullah, Christopher D. McGann, Diana Velez, Ying S. Ting, Asaf Poran, Daniel A. Rothenberg, Sagar Chhangawala, Alex Rubinsteyn, Jeff Hammerbacher, Richard B. Gaynor, Edward F. Fritsch, Rob C. Oslund, Dominik Barthelme, Terri A. Addona, Christina M. Arieta, Michael S. Rooney. Enhanced HLA-II epitope prediction for immunotherapy with novel proteomics and genomics approaches [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B23.

Cancer genome sequencing has enabled the rapid identification of the complete repertoire of coding sequence mutations within a patient's tumor and facilitated their use as personalized immunogens. Although a variety of techniques are available to assist in the selection of mutation-defined epitopes to be included within the tumor vaccine, the ability of the peptide to bind to patient MHC is a key gateway to peptide presentation. With advances in the accuracy of predictive algorithms for MHC class I binding, choosing epitopes on the basis of predicted affinity provides a rapid and unbiased approach to epitope prioritization. We show herein the retrospective application of a prediction algorithm to a large set of bona fide T cell–defined mutated human tumor antigens that induced immune responses, most of which were associated with tumor regression or long-term disease stability. The results support the application of this approach for epitope selection and reveal informative features of these naturally occurring epitopes to aid in epitope prioritization for use in tumor vaccines. Cancer Immunol Res; 2(6); 522–9. ©2014 AACR.

Despite years of preclinical efforts and hundreds of clinical studies, therapeutic cancer vaccines with the routine ability to limit or eliminate tumor growth in humans have been elusive. With advances in genome sequencing, it is now possible to identify a new class of tumor-specific antigens derived from mutated proteins that are present only in the tumor. These “neoantigens” should provide highly specific targets for antitumor immunity. Although many challenges remain in producing and testing neoantigen-based vaccines customized for each patient, a neoantigen vaccine offers a promising new approach to induce highly focused antitumor T cells aimed at eradicating cancer cells. Cancer Immunol Res; 1(1); 11–15. ©2013 AACR.

BACKGROUND: The impact of individualized neoepitope vaccination targeting neoantigens arising from tumor-specific mutations for GBM, a low mutation burden tumor with an immunologically cold tumor microenvironment, as well as that of concurrently administered dexamethasone (dex), are unknown. METHODS: Individualized vaccination of up to 20 synthetic, long neoepitope peptides with high predicted HLA binding affinity admixed with poly-ICLC, were administered subcutaneously using a prime-boost schedule after RT to newly diagnosed, MGMT unmethylated, at least partially resected, GBM patients without progression after radiation (RT) in our phase 1b study. RESULTS: 9 of 10 screened patients had sufficient (10) identified neoepitope peptides. 8 patients without progression after RT received vaccine consisting of a median of 12 peptides (range, 7–20) beginning a median of 19.9 wks (range 17.1–24.7) after surgery. Adverse events were limited to infrequent grade 1/2 local reactions and fatigue. Median PFS and OS were 7.5 mths (90% CI: 6.2, 9.7) and 16.8 mths (90% CI: 9.6, 21.3). Evaluation of neoepitope-specific immune responses and tumor immune infiltrate analyses were performed on five patients with pre- and post-vaccination samples. Three patients on dex for post-RT edema during vaccine had no immune responses and no change in tumor infiltrating effector cells. In contrast 2 patients not on dex had robust, de novo immune responses against multiple predicted personal neoantigens including polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses that were enriched for memory and activated phenotypes as well as increased numbers of tumor-infiltrating CD4+ and CD8+ T cells. T cell receptor analysis from one patient identified identical clonotypes isolated from post-vaccination tumor tissue and peripheral blood including two clonotypes specific for ARHGAP35, a neoantigen targeted by vaccination. CONCLUSIONS: Individualized, multi-neoepitope vaccines are feasible, safe and capable of generating systemic and intra-tumoral immune responses in GBM patients that appear to be abrogated by dex.

2020Background: The impact of individualized neoepitope vaccination targeting neoantigens arising from tumor-specific mutations for GBM, a low mutation burden tumor with an immunologically cold tum...

Nature 547, 217–221 (2017); doi:10.1038/nature22991 In this Letter, the ‘Data availability’ section in the Methods should state ‘WES and RNA-seq data are deposited in dbGaP (https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs001451.v1.p1). All other data are available from the corresponding author upon reasonable request.

Genome sequencing has revealed a large number of shared and personal somatic mutations across human cancers. In principle, any genetic alteration affecting a protein-coding region has the potential to generate mutated peptides that are presented by surface HLA class I proteins that might be recognized by cytotoxic T cells. To test this possibility, we implemented a streamlined approach for the prediction and validation of such neoantigens derived from individual tumors and presented by patient-specific HLA alleles. We applied our computational pipeline to 91 chronic lymphocytic leukemias (CLLs) that underwent whole-exome sequencing (WES). We predicted ∼22 mutated HLA-binding peptides per leukemia (derived from ∼16 missense mutations) and experimentally confirmed HLA binding for ∼55% of such peptides. Two CLL patients that achieved long-term remission following allogeneic hematopoietic stem cell transplantation were monitored for CD8(+) T-cell responses against predicted or confirmed HLA-binding peptides. Long-lived cytotoxic T-cell responses were detected against peptides generated from personal tumor mutations in ALMS1, C6ORF89, and FNDC3B presented on tumor cells. Finally, we applied our computational pipeline to WES data (N = 2488 samples) across 13 different cancer types and estimated dozens to thousands of predicted neoantigens per individual tumor, suggesting that neoantigens are frequent in most tumors.

Neoantigen-based cancer vaccines designed to target the unique immunogenic mutations arising in each patient’s tumor are breathing new life into a struggling approach. Data continue to demonstrate the importance of neoantigens in immune control of cancer. Despite manufacturing complexity, outstanding questions and desired further improvements, neoantigen vaccines are currently undergoing clinical evaluation.

Stimulating the production of fetal hemoglobin may benefit patients with sickle cell anemia by inhibiting sickling. We gave pulsed treatments with high doses of recombinant human erythropoietin to baboons in order to test the hypothesis that the resultant rapid erythroid regeneration would stimulate F cells--i.e., cells that contain fetal hemoglobin. In normal animals, this treatment caused sharp increments in F-reticulocyte levels, which rose from 1 to 2 percent before treatment to 40 to 50 percent afterward. In two animals with chronic anemia and high levels of endogenous erythropoietin, recombinant human erythropoietin induced further increments in F-reticulocyte levels, which rose in one animal from 6 to 8 percent before treatment to 23 percent after treatment, and in the other from 20 percent before to 50 percent afterward. The time course of F-reticulocyte stimulation suggested that these cells were the products of mobilized early erythroid progenitors. These results raise the possibility that pulses of erythropoietin could be used to stimulate F-cell formation in patients with sickle cell disease.

A cloned library of large, random embryonic human DNA fragments was constructed and screened for beta-globin sequences using the cloned human beta-globin cDNA plasmid pJW102 (Wilson et al., 1978) as a hybridization probe. Two independent clones were obtained and then characterized by restriction endonuclease cleavage analysis, hybridization experiments and partial DNA sequencing. Each of the clones carries both the adult delta- and beta-globin genes. The two genes are separated by approximately 5.4 kilobases (kb) of DNA and their orientation with respect to the direction of transcription is 5'-delta--beta-3'. Both the delta- and beta-globin genes contain a large noncoding intervening sequence (950 and 900 bp, respectively) located between the codons for amino acids 104 (arginine) and 105 (leucine). Although the location of the large intervening sequence within the coding regions of the two genes is identical, the two noncoding sequences bear little sequence homology. A second, smaller intervening sequence similar to that found in other mammalian beta-globin genes was detected near the 5' end of the human beta-globin gene. The two independently isolated beta-globin clones differ from each other by the presence of a Pst I restriction enzyme cleavage site within the large intervening sequence of the delta-globin gene of one of the clones. This suggests that the human DNA carried in the two clones was derived from two homologous chromosomes which were heterozygous for the Pst I restriction enzyme recognition sequence.

Chronically anemic baboons on a continuous hydroxyurea regimen were treated with pulsed doses of recombinant human erythropoietin (rHuEpo) to test whether the combination of these two compounds, which individually induce F-cell production, can enhance further F-cell output. A low-F-cell-responding animal under chronic hydroxyurea treatment was given three separate pulses of Epo and responded with F- reticulocyte increments that were similar to the sum increments caused by either hydroxyurea alone or rHuEpo alone. The same results were obtained in a high-F-responding animal similarly treated. These findings suggest that rHuEpo and hydroxyurea can increase F cell numbers in an additive fashion. It is speculated that both compounds act through perturbation of erythroid differentiation kinetics.

INTRODUCTION 146 The Ontogeny of Globin Gene Expression 146 Globin Gene Mapping and Molecular Cloning 147 THE HUMAN ,8-LIKE GLOBIN GENES .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . .. . .. . . . . 148 ,8-Like Globin Gene Fine Structure 148 ,8-Like Globin Gene Linkage . . . .... 151 Repetitive Sequence Elements Within Globin Gene Clusters 153 ,8-Globin Variants .... 153 DNA Sequence Polymorphisms Within the ,8-Like Globin Gene Cluster ..... 154

The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.

Deletions in the DNA of individuals with hereditary persistence of fetal haemoglobin (HPFH) and 8 beta-thalassaemia have been mapped as a means of identifying regulatory sequences involved in the switch from fetal to adult globin gene expression. The end points of these deletions have been precisely located with respect to restriction endonuclease cleavage sites within and surrounding the gamma-, delta- and beta-globin genes in normal human DNA and the deletion maps were used to obtain definitive evidence for the physical linkage of the fetal and adult beta-like globin genes in the order 5'Ggamma-Agamma-delta-beta 3'. Correlation of haematological data and the location of deletions in two cases of HPFH and one case of deltabeta-thalassaemia suggest that a region of DNA located near the 5'-end of the delta-globin gene may be involved in the suppression in cis of gamma-globin gene expression in adults. The interpretation of a second case of deltabeta-thalassaemia is complicated by the fact that the deletion removes the Agamma-gene in addition to the region near the 5'-end of the delta-globin gene.