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Koelle, D. M.

Publications and source records attributed to Koelle, D. M..

11 recordsLinked to original sources

Heterogeneity of HCMV-Specific CD8+ T Cells is Established Early in Infection, Clonally Encoded, and Stable Over Time

Human cytomegalovirus (CMV) elicits lifelong memory T cell responses of striking size and phenotypic diversity, but how these diverse responses are established during acute infection and evolve over decades of chronic stimulation is poorly understood. We tracked CD8+ T cells specific for the viral antigens pp65 and IE1 by single-cell RNAseq, TCRseq, and CITEseq over a period of 16 months in a newly seroconverted donor, and in five latently infected donors over intervals ranging from 4.5 to 12.5 years. We found that individual T cell clones exhibited distinct, specificity-dependent transcriptional profiles, with pp65-specific cells skewed toward central memory phenotypes and IE1-specific cells enriched for effector and chronically activated states. Among pp65-specific T cells we identified a CMC1-expressing state not previously described in virus-specific T cells that shares transcriptional features with exhausted T cells in human tumors yet remains phenotypically stable and clonally intact for over a decade of viral latency. Distinct clonal phenotypes arose during acute infection and were remarkably stable during viral latency, and these phenotypes tracked with relative TCR avidity and TCR sequence. Our findings reveal that the phenotypic diversity of the human CMV-specific CD8+ T cell response is organized at the level of individual clones, with distinct functional identities established during acute infection and preserved with striking fidelity across decades of viral latency and chronic antigen exposure.

immunology↗

Cytotoxic T cells targeting lytic KSHV gene products infiltrate Kaposi sarcoma tumors

Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and KSHV-associated multicentric Castlemans disease (MCD), malignancies that predominantly arise in the context of T-cell deficiency. Unlike responses to other human herpesviruses such as EBV and CMV, KSHV-specific T-cell responses detected in blood have been described as heterogeneous and low-intensity. Hypothesizing that KSHV-specific T cells are recruited to KS tumors, we analyzed the T-cell receptor (TCR) repertoire of biopsies from 144 Ugandan adults with KS (106 people living with HIV [PLWH], 38 HIV-seronegative) and identified >4,000 {beta} TCRs with predicted specificity for KSHV- or HIV-encoded peptides presented by specific MHC alleles. We tested 14 putative KSHV- or HIV-specific TCRs for recognition of cells presenting cognate peptides in the predicted MHC context. Three novel HIV-specific TCRs, found only in tumors from PLWH, exhibited high-avidity, MHC-restricted recognition of HIV Vpr and Nef peptides previously identified as CD8+ T-cell targets. Four KSHV-specific TCRs, detected in tumors from both PLWH and HIV-seronegative individuals, recognized peptides encoded by the lytic KSHV genes ORF6, ORF57, and ORF59. The ORF6- and ORF57-specific TCRs were observed in multiple individuals and constitute the first examples of public T-cell responses to KSHV. We then confirmed that these four KSHV-specific TCRs recognized KSHV-infected cells undergoing lytic reactivation. Identification of TCRs specific for KSHV lytic gene products will enable the development of T-cell-based therapies for KS and other KSHV-associated diseases. Author summaryKaposi sarcoma-associated herpesvirus (KSHV) is the only oncogenic human virus for which there is no effective vaccine, antiviral, or immunotherapeutic strategy that directly targets the virus. KSHV is the causative agent of Kaposi sarcoma and primary effusion lymphoma, cancers that primarily affect immunocompromised individuals, most commonly people living with HIV in sub-Saharan Africa, where KSHV is endemic and HIV infection is prevalent. In this study we defined the antigenic specificity of novel, putative KSHV- and HIV-specific T-cell receptors (TCRs) that are carried in T cells commonly infiltrating KS tumor biopsies. We show that T cells engineered to express KSHV-specific TCRs exhibit high-avidity, MHC-restricted recognition of KSHV-infected cells presenting naturally processed peptides encoded by KSHV lytic genes. Our findings provide a blueprint for dissecting KSHV-specific T-cell immunity and advancing the development of immunotherapeutic strategies for the prevention or treatment of KSHV-associated diseases.

immunology↗

B cells specific for polyomavirus-derived oncoprotein are predictive of Merkel cell carcinoma progression

Merkel cell carcinomas typically arise from clonal integration of the Merkel cell polyomavirus. Immunogenic viral oncoproteins then lead to tumorigenesis. Oncoprotein-specific T cells are essential for anti-MCC immunity, but it is unclear whether B cells promote tumor control. Here, we analyzed the frequency and phenotype of viral oncoprotein-specific and total B cells in 47 blood samples and 19 unmatched tumors from MCC patients-- of which 8 out 19 progressed. The phenotype of blood B cells did not correlate with MCC patient outcomes. In contrast, all 11 patients with robust oncoprotein-specific antibody-secreting and/or germinal center B cells in tumors experienced long-term MCC control. In vitro, B cells engineered to be specific for viral oncoproteins increased the sensitivity of oncoprotein-specific CD4+ T cells by over 50-fold. Together, our findings suggest that cancer-specific B cells promote anti-tumor immunity via increased T cell responses and that cancer-specific B cell augmentation could be therapeutically relevant. Statement of SignificanceThe link between cancer-specific B cells in anti-tumor immunity and clinical outcomes remains poorly defined. Here, we show that tumor-associated B cells specific for a viral oncoprotein expressed in MCC patient tumors predict disease control with remarkable accuracy, establishing their potential as active participants in tumor immunity.

immunology↗

TCR2HLA: calibrated inference of HLA genotypes from TCR repertoires enables identification of immunologically relevant metaclonotypes

T cell receptors (TCRs) recognize peptides presented by polymorphic human leukocyte antigen (HLA) molecules, but HLA genotype data are often missing from TCR repertoire sequencing studies. To address this, we developed TCR2HLA, an open-source tool that infers HLA genotypes from TCR{beta} repertoires. Expanding on work linking public TRBV-CDR3 sequences to HLA genotypes, we incorporated "quasi-public" metaclonotypes - composed of rarer TCR{beta} sequences with shared amino acid features - enriched by HLA genotypes. Using four TCR{beta}seq datasets from 3,150 individuals, we applied TRBV gene partitioning and locality-sensitive hashing to identify [~]96,000 TCR{beta} features strongly associated with specific HLA alleles from 71M input TCRs. Binary HLA classifiers built with these features achieved high balanced accuracy (>0.9) across common HLA-A (9/12), B (9/12), C (6/13), DRB1 (11/11) alleles and prevalent DPA1/DPB1 (6/10), DQA1/DQB1 (8/17) heterodimers. We also introduced a high-sensitivity calibration to support predictions in samples with as few as 5,000 unique clonotypes. Calibrated predictions with confidence filtering improved reliability. Beyond genotype imputation, TCR2HLA enables the discovery of novel HLA- and exposure-associated TCRs, as shown by the identification of SARS-CoV-2 related TCRs in a large COVID-19 dataset lacking HLA data. TCR2HLA provides a scalable framework for bridging the gap between TCRseq data and HLA genotype for biomarker discovery.

immunology↗

Treponema pallidum periplasmic and membrane proteins are recognized by circulating and skin CD4+ T cells

BackgroundHistologic and serologic studies suggest the induction of local and systemic Treponema pallidum (Tp)-specific CD4+ T cell responses to Tp infection. We hypothesized that Tp-specific CD4+ T cells are detectable in blood and in the skin rash of secondary syphilis and persist in both compartments after treatment. MethodsPBMC collected from 67 participants were screened by IFN{gamma} ELISPOT response to Tp sonicate. Tp-reactive T cell lines from blood and skin were probed for responses to 88 recombinant Tp antigens. Peptide epitopes and HLA class II restriction were defined for selected antigens. ResultsWe detected CD4+ T cell responses to Tp sonicate ex vivo. Using Tp-reactive T cell lines we observed recognition of 14 discrete proteins, 13 of which localize to bacterial membranes or the periplasmic space. After therapy, Tp-specific T cells persisted for at least 6 months in skin and 10 years in blood. ConclusionsTp infection elicits an antigen-specific CD4+ T cell response in blood and skin. Tp-specific CD4+ T cells persist as memory in both compartments long after curative therapy. The Tp antigenic targets we identified may be high priority vaccine candidates.

immunology↗

Reanalysis of single-cell RNA sequencing data does not support herpes simplex virus 1 latency in non-neuronal ganglionic cells in mice

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1) and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent virus is also present in immune cells recovered from ganglia in a mouse model used for studying latency. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for this conclusion. Unexpectedly, off-target priming in 3 scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in a mixed population of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained by inaccuracies in the data analyses.

microbiology↗

CD4 and CD8 co-receptors modulate functional avidity of CD1b-restricted T cells.

CD4 and CD8 co-receptors define distinct lineages of T cells restricted by major histocompatibility complex (MHC) Class II and I molecules, respectively. Co-receptors interact with the T cell receptor (TCR) at the surface of MHC-restricted T cells to facilitate antigen recognition, thymic selection, and functional differentiation. T cells also recognize lipid antigens presented by CD1 molecules, but the role that CD4 and CD8 play in lipid antigen recognition is unknown. We studied the effect of CD4 and CD8 on the avidity, activation, and function of T cells specific for two CD1b-presented mycobacterial lipid antigens, glucose monomycolate (GMM) and diacylated sulfoglycolipids (SGL). In a human cohort study using SGL-loaded CD1b tetramers, we discovered a hierarchy among SGL-specific T cells in which T cells expressing the CD4 or CD8 co-receptor stain with a higher tetramer mean fluorescence intensity (MFI) than CD4-CD8- T cells. To determine the role of the TCR co-receptor in lipid antigen recognition, we exogenously expressed GMM and SGL-specific TCRs in Jurkat or polyclonal T cells and quantified tetramer staining and activation thresholds. Transduced CD4+ primary T cells bound the lipid-loaded CD1b tetramer with a higher MFI than CD8+ primary T cells, and transduced CD8+ Jurkat cells bound the SGL-CD1b tetramer with higher MFI than CD4-CD8- Jurkat cells. The presence of either co-receptor also decreased the threshold for IFN-{gamma} secretion. Further, co-receptor expression increased surface expression of CD3{varepsilon}, suggesting a mechanism for increased tetramer binding and activation. Finally, we used single-cell sequencing to define the TCR repertoire and ex vivo functional profiles of SGL-specific T cells from individuals with M.tb disease. We found that CD8+ T cells specific for SGL express canonical markers associated with cytotoxic T lymphocytes, while CD4+ T cells could be classified as T regulatory or T follicular helper cells. Among SGL-specific T cells, only those expressing the CD4 co-receptor also expressed Ki67, suggesting that they were actively proliferating at the time of sample collection. Together, these data reveal that expression of CD4 and CD8 co-receptor modulates TCR avidity for lipid antigen, leading to functional diversity and differences in in vivo proliferation during M.tb disease.

immunology↗

In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N=80 samples from persons with RT-PCR confirmed SARS-CoV2 infection), and a specificity of 97.2% (N=106 control samples).

bioinformatics↗

The impact of interspecies recombination on human herpes simplex virus evolution and host immune recognition

Among the most ubiquitous of human pathogens, HSV-1 and HSV-2 are distinct viral species that diverged about six million years ago. At least four ancient HSV-1 x HSV-2 interspecies recombination events have affected the HSV-2 genome, with recombinants and non-recombinants at each locus circulating today. Though interspecies recombination has occurred in the past, its importance in HSV evolution remains incompletely defined. Using 255 newly-sequenced and 219 existing HSV genome sequences, we comprehensively assessed interspecies recombination in HSV. The novel recombinants we identify demonstrate that the sizes and locations of interspecies recombination events in HSV-2 are more variable than previously appreciated. One novel recombinant arose in its current host, showing for the first time that interspecies recombination occurs in contemporary HSV populations. We also demonstrate that interspecies recombination affects T-cell recognition of HSV. Our findings indicate that interspecies recombination can significantly influence genetic variation in and host immunologic response to HSV-2.

genomics↗

Ultrasensitive capture of human herpes simplex virus genomes directly from clinical samples reveals extraordinarily limited evolution in cell culture

Herpes simplex viruses (HSV) are difficult to sequence due to their large DNA genome, high GC content, and the presence of repeats. To date, most HSV genomes have been recovered from culture isolates, raising concern that these genomes may not accurately represent circulating clinical strains. We report the development and validation of a DNA oligonucleotide hybridization panel to recover near complete HSV genomes at abundances up to 50,000-fold lower than previously reported. Using copy number information on herpesvirus and host DNA background via quantitative PCR, we developed a protocol for pooling for cost-effective recovery of more than 50 HSV-1 or HSV-2 genomes per MiSeq run. We demonstrate the ability to recover >99% of the HSV genome at >100X coverage in 72 hours at viral loads that allow whole genome recovery from latently-infected ganglia. We also report a new computational pipeline for rapid HSV genome assembly and annotation. Using the above tools and a series of 17 HSV-1-positive clinical swabs sent to our laboratory for viral isolation, we show limited evolution of HSV-1 during viral isolation in human fibroblast cells compared to the original clinical samples. Our data indicate that previous studies using low passage clinical isolates of herpes simplex viruses are reflective of the viral sequences present in the lesion and thus can be used in phylogenetic analyses. We also detect superinfection within a single sample with unrelated HSV-1 strains recovered from separate oral lesions in an immunosuppressed patient during a 2.5-week period, illustrating the power of direct-from-specimen sequencing of HSV.\n\nImportanceHerpes simplex viruses affect more than 4 billion people across the globe, constituting a large burden of disease. Understanding global diversity of herpes simplex viruses is important for diagnostics and therapeutics as well as cure research and tracking transmission among humans. To date, most HSV genomics has been performed on culture isolates and DNA swabs with high quantities of virus. We describe the development of wet-lab and computational tools that enable the accurate sequencing of near-complete genomes of HSV-1 and HSV-2 directly from clinical specimens at abundances >50,000-fold lower than previously sequenced and at significantly reduced cost. We use these tools to profile circulating HSV-1 strains in the community and illustrate limited changes to the viral genome during the viral isolation process. These techniques enable cost-effective, rapid sequencing of HSV-1 and HSV-2 genomes that will help enable improved detection, surveillance, and control of this human pathogen.

genomics↗

Copy number heterogeneity, large origin tandem repeats, and interspecies recombination in HHV-6A and HHV-6B reference strains

Quantitative PCR is the diagnostic pillar for clinical virology testing, and reference materials are necessary for accurate, comparable quantitation between clinical laboratories. Accurate quantitation of HHV-6 is important for detection of viral reactivation and inherited chromosomally integrated HHV-6 in immunocompromised patients. Reference materials in clinical virology commonly consist of laboratory-adapted viral strains that may be affected by the culture process. We performed next-generation sequencing to make relative copy number measurements at single nucleotide resolution of eight candidate HHV-6A and seven HHV-6B reference strains and DNA materials from the HHV-6 Foundation and Advanced Biotechnologies. 11 of 17 (65%) HHV6 candidate reference materials showed multiple copies of the origin of replication upstream of the U41 gene by next-generation sequencing. These large tandem repeats arose independently in culture-adapted HHV-6A and HHV-6B strains, measuring 1254 bp and 983 bp, respectively. Copy number measured between 4-10X copies relative to the rest of the genome. We also report the first interspecies recombinant HHV-6 strain with a HHV-6A GS backbone and >5.5kb region from HHV-6B Z29 from U41-U43 that covered the origin tandem repeat. Specific HHV-6A reference strains demonstrated duplication of regions at UL1/UL2, U87, and U89, as well as deletion in the U12-U24 region and U94/95 genes. HHV-6 strains derived from cord blood mononuclear cells from different labs on different continents revealed no copy number differences throughout the viral genome. These data indicate large origin tandem duplications are an adaptation of both HHV-6A and HHV-6B in culture and show interspecies recombination is possible within the Betaherpesvirinae.\n\nImportanceAnything in science that needs to be quantitated requires a standard unit of measurement. This includes viruses, for which quantitation increasingly determines definitions of pathology and guidelines for treatment. However, the act of making standard or reference material in virology can alter its very usefulness through genomic duplications, insertions, and rearrangements. We used deep sequencing to examine candidate reference strains for HHV-6, a ubiquitous human virus that can reactivate in the immunocompromised population and is integrated into the human genome in every cell of the body for 1% of people worldwide. We found large tandem repeats in the origin of replication for both HHV-6A and HHV-6B that are selected for in culture. We also found the first interspecies recombinant between HHV-6A and HHV-6B, a phenomenon that is well-known in alphaherpesviruses but to date has not been seen in betaherpesviruses. These data critically inform HHV-6 biology and the standard selection process.

genomics↗