Inhibition of programmed death-ligand 1 (PD-L1) with atezolizumab can induce durable clinical benefit (DCB) in patients with metastatic urothelial cancers, including complete remissions in patients ...with chemotherapy refractory disease. Although mutation load and PD-L1 immune cell (IC) staining have been associated with response, they lack sufficient sensitivity and specificity for clinical use. Thus, there is a need to evaluate the peripheral blood immune environment and to conduct detailed analyses of mutation load, predicted neoantigens, and immune cellular infiltration in tumors to enhance our understanding of the biologic underpinnings of response and resistance.
The goals of this study were to (1) evaluate the association of mutation load and predicted neoantigen load with therapeutic benefit and (2) determine whether intratumoral and peripheral blood T cell receptor (TCR) clonality inform clinical outcomes in urothelial carcinoma treated with atezolizumab. We hypothesized that an elevated mutation load in combination with T cell clonal dominance among intratumoral lymphocytes prior to treatment or among peripheral T cells after treatment would be associated with effective tumor control upon treatment with anti-PD-L1 therapy. We performed whole exome sequencing (WES), RNA sequencing (RNA-seq), and T cell receptor sequencing (TCR-seq) of pretreatment tumor samples as well as TCR-seq of matched, serially collected peripheral blood, collected before and after treatment with atezolizumab. These parameters were assessed for correlation with DCB (defined as progression-free survival PFS >6 months), PFS, and overall survival (OS), both alone and in the context of clinical and intratumoral parameters known to be predictive of survival in this disease state. Patients with DCB displayed a higher proportion of tumor-infiltrating T lymphocytes (TIL) (n = 24, Mann-Whitney p = 0.047). Pretreatment peripheral blood TCR clonality below the median was associated with improved PFS (n = 29, log-rank p = 0.048) and OS (n = 29, log-rank p = 0.011). Patients with DCB also demonstrated more substantial expansion of tumor-associated TCR clones in the peripheral blood 3 weeks after starting treatment (n = 22, Mann-Whitney p = 0.022). The combination of high pretreatment peripheral blood TCR clonality with elevated PD-L1 IC staining in tumor tissue was strongly associated with poor clinical outcomes (n = 10, hazard ratio (HR) (mean) = 89.88, HR (median) = 23.41, 95% CI 2.43, 506.94, p(HR > 1) = 0.0014). Marked variations in mutation loads were seen with different somatic variant calling methodologies, which, in turn, impacted associations with clinical outcomes. Missense mutation load, predicted neoantigen load, and expressed neoantigen load did not demonstrate significant association with DCB (n = 25, Mann-Whitney p = 0.22, n = 25, Mann-Whitney p = 0.55, and n = 25, Mann-Whitney p = 0.29, respectively). Instead, we found evidence of time-varying effects of somatic mutation load on PFS in this cohort (n = 25, p = 0.044). A limitation of our study is its small sample size (n = 29), a subset of the patients treated on IMvigor 210 (NCT02108652). Given the number of exploratory analyses performed, we intend for these results to be hypothesis-generating.
These results demonstrate the complex nature of immune response to checkpoint blockade and the compelling need for greater interrogation and data integration of both host and tumor factors. Incorporating these variables in prospective studies will facilitate identification and treatment of resistant patients.
The vast diversity of B-cell receptors (BCR) and secreted antibodies enables the recognition of, and response to, a wide range of epitopes, but this diversity has also limited our understanding of ...humoral immunity. We present a public database of more than 37 million unique BCR sequences from three healthy adult donors that is many fold deeper than any existing resource, together with a set of online tools designed to facilitate the visualization and analysis of the annotated data. We estimate the clonal diversity of the naive and memory B-cell repertoires of healthy individuals, and provide a set of examples that illustrate the utility of the database, including several views of the basic properties of immunoglobulin heavy chain sequences, such as rearrangement length, subunit usage, and somatic hypermutation positions and dynamics.
An individual's T cell repertoire dynamically encodes their pathogen exposure history. To determine whether pathogen exposure signatures can be identified by documenting public T cell receptors ...(TCRs), we profiled the T cell repertoire of 666 subjects with known cytomegalovirus (CMV) serostatus by immunosequencing. We developed a statistical classification framework that could diagnose CMV status from the resulting catalog of TCRβ sequences with high specificity and sensitivity in both the original cohort and a validation cohort of 120 different subjects. We also confirmed that three of the identified CMV-associated TCRβ molecules bind CMV in vitro, and, moreover, we used this approach to accurately predict the HLA-A and HLA-B alleles of most subjects in the first cohort. As all memory T cell responses are encoded in the common format of somatic TCR recombination, our approach could potentially be generalized to a wide variety of disease states, as well as other immunological phenotypes, as a highly parallelizable diagnostic strategy.
The human adaptive immune system must generate extraordinary diversity to be able to respond to all possible pathogens. The T-cell repertoire derives this high diversity through somatic recombination ...of the T-cell receptor (TCR) locus, a random process that results in repertoires that are largely private to each individual. However, factors such as thymic selection and T-cell proliferation upon antigen exposure can affect TCR sharing among individuals. By immunosequencing the TCRβ variable region of 426 healthy individuals, we find that, on average, fewer than 1% of TCRβ clones are shared between individuals, consistent with largely private TCRβ repertoires. However, we detect a significant correlation between increased HLA allele sharing and increased number of shared TCRβ clones, with each additional shared HLA allele contributing to an increase in ~0.01% of the total shared TCRβ clones, supporting a key role for HLA type in shaping the immune repertoire. Surprisingly, we find that shared antigen exposure to CMV leads to fewer shared TCRβ clones, even after controlling for HLA, indicative of a largely private response to major viral antigenic exposure. Consistent with this hypothesis, we find that increased age is correlated with decreased overall TCRβ clone sharing, indicating that the pattern of private TCRβ clonal expansion is a general feature of the T-cell response to other infectious antigens as well. However, increased age also correlates with increased sharing among the lowest frequency clones, consistent with decreased repertoire diversity in older individuals. Together, all of these factors contribute to shaping the TCRβ repertoire, and understanding their interplay has important implications for the use of T cells for therapeutics and diagnostics.
Radiotherapy is a highly effective anticancer treatment forming part of the standard of care for the majority of patients, but local and distal disease recurrence remains a major cause of mortality. ...Radiotherapy is known to enhance tumor immunogenicity; however, the contribution and mechanisms of radiotherapy-induced immune responses are unknown.
The impact of low-dose fractionated radiotherapy (5 × 2 Gy) alone and in combination with αPD-1 mAb on the tumor microenvironment was evaluated by flow cytometry and next-generation sequencing of the T-cell receptor (TCR) repertoire. A dual-tumor model was used, with fractionated radiotherapy delivered to a single tumor site to enable evaluation of the local and systemic response to treatment and ability to induce abscopal responses outside the radiation field.
We show that fractionated radiotherapy leads to T-cell infiltration at the irradiated site; however, the TCR landscape remains dominated by polyclonal expansion of preexisting T-cell clones. Adaptive resistance via the PD-1/PD-L1 pathway restricts the generation of systemic anticancer immunity following radiotherapy, which can be overcome through combination with αPD-1 mAb leading to improved local and distal tumor control. Moreover, we show that effective clearance of tumor following combination therapy is dependent on both T cells resident in the tumor at the time of radiotherapy and infiltrating T cells.
These data provide evidence that radiotherapy can enhance T-cell trafficking to locally treated tumor sites and augment preexisting anticancer T-cell responses with the capacity to mediate regression of out-of-field tumor lesions when delivered in combination with αPD-1 mAb therapy.
.
Recognized clinical risk factors for progression (emergency presentation, poorly differentiated tumor, depth of tumor invasion, and adjacent organ involvement) are insufficient to identify those ...patients with stage II CRC at higher risk of disease progression 4,5. ...a meta-analysis aimed to assess the predictive ability of these signatures revealed that although gene expression signatures may be associated with prognosis, their ability to accurately predict patients’ risk of progression was limited, probably due to the molecular heterogeneity of tumors 7. ...the identification of new biomarkers to inform clinical decision-making for adjuvant chemotherapy is needed 8. Methods Patients and samples The discovery dataset (named ICO/CLX) included a previously described set of 100 patients with colon cancer diagnosed at stage II and MSS paired normal-tumor samples (Colonomics study, “CLX”: www.colonomics.org; NCBI BioProject PRJNA188510). Raw sequence data were filtered based on the TCRβ V, D, and J gene definitions provided by the international ImMunoGeneTics information system (IMGT) database and binned using a modified nearest-neighbor algorithm to merge closely related sequences and remove both PCR and sequencing errors.
The Ad26.COV2.S vaccine
has demonstrated clinical efficacy against symptomatic COVID-19, including against the B.1.351 variant that is partially resistant to neutralizing antibodies
. However, the ...immunogenicity of this vaccine in humans against SARS-CoV-2 variants of concern remains unclear. Here we report humoral and cellular immune responses from 20 Ad26.COV2.S vaccinated individuals from the COV1001 phase I-IIa clinical trial
against the original SARS-CoV-2 strain WA1/2020 as well as against the B.1.1.7, CAL.20C, P.1 and B.1.351 variants of concern. Ad26.COV2.S induced median pseudovirus neutralizing antibody titres that were 5.0-fold and 3.3-fold lower against the B.1.351 and P.1 variants, respectively, as compared with WA1/2020 on day 71 after vaccination. Median binding antibody titres were 2.9-fold and 2.7-fold lower against the B.1.351 and P.1 variants, respectively, as compared with WA1/2020. Antibody-dependent cellular phagocytosis, complement deposition and natural killer cell activation responses were largely preserved against the B.1.351 variant. CD8 and CD4 T cell responses, including central and effector memory responses, were comparable among the WA1/2020, B.1.1.7, B.1.351, P.1 and CAL.20C variants. These data show that neutralizing antibody responses induced by Ad26.COV2.S were reduced against the B.1.351 and P.1 variants, but functional non-neutralizing antibody responses and T cell responses were largely preserved against SARS-CoV-2 variants. These findings have implications for vaccine protection against SARS-CoV-2 variants of concern.
In psoriasis, an IL-17-mediated inflammatory skin disease, skin lesions resolve with therapy, but often recur in the same locations when therapy is discontinued. We propose that residual T cell ...populations in resolved psoriatic lesions represent the pathogenic T cells of origin in this disease. Utilizing high-throughput screening (HTS) of the T cell receptor (TCR) and immunostaining, we found that clinically resolved psoriatic lesions contained oligoclonal populations of T cells that produced IL-17A in both resolved and active psoriatic lesions. Putative pathogenic clones preferentially utilized particular Vβ and Vα subfamilies. We identified 15 TCRβ and 4 TCRα antigen receptor sequences shared between psoriasis patients and not observed in healthy controls or other inflammatory skin conditions. To address the relative roles of αβ versus γδ T cells in psoriasis, we carried out TCR/δ HTS. These studies demonstrated that the majority of T cells in psoriasis and healthy skin are αβ T cells. γδ T cells made up 1% of T cells in active psoriasis, less than 1% in resolved psoriatic lesions, and less than 2% in healthy skin. All of the 70 most frequent putative pathogenic T cell clones were αβ T cells. In summary, IL-17-producing αβ T cell clones with psoriasis-specific antigen receptors exist in clinically resolved psoriatic skin lesions. These cells likely represent the disease-initiating pathogenic T cells in psoriasis, suggesting that lasting control of this disease will require suppression of these resident T cell populations.
To understand better how selection processes balance the benefits of Ig repertoire diversity with the risks of autoreactivity and nonfunctionality of highly variable IgH CDR3s, we collected millions ...of rearranged germline IgH CDR3 sequences by deep sequencing of DNA from mature human naive B cells purified from four individuals and analyzed the data with computational methods. Long HCDR3 regions, often components of HIV-neutralizing Abs, appear to derive not only from incorporation of long D genes and insertion of large N regions but also by usage of multiple D gene segments in tandem. However, comparison of productive and out-of-frame IgH rearrangements revealed a selection bias against long HCDR3 loops, suggesting these may be disproportionately either poorly functional or autoreactive. Our data suggest that developmental selection removes HCDR3 loops containing patches of hydrophobicity, which are commonly found in some auto-antibodies, and at least 69% of the initial productive IgH rearrangements are removed from the repertoire during B cell development. Additionally, we have demonstrated the potential utility of this new technology for vaccine development with the identification in all four individuals of related candidate germline IgH precursors of the HIV-neutralizing Ab 4E10.
The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and ...IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines.
Sensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay's ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels.
LoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low.
These studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.
PDF
