Circulating T-Cell Repertoires Correlate With the Tumor Response in Patients With Breast Cancer Receiving Neoadjuvant Chemotherapy

PURPOSE Neoadjuvant chemotherapy (NAC) has been widely used in patients with breast cancer to minish tumor burden and increase resection rate of cancer. T-cell repertoire has been believed to be able to monitor antitumor immune responses. This study aimed to explore the dynamic change of T-cell repertoire and its clinical value in evaluating the tumor response in patients with breast cancer receiving NAC. MATERIALS AND METHODS Ninety-four patients who underwent NAC before surgery were recruited, and peripheral blood samples were collected at multiple time points during NAC. High-throughput T-cell receptor (TCR)-β sequencing was used to characterize the T-cell repertoire of every sample and analyzed the changes in circulating T-cell repertoire during NAC. RESULTS We found that the diversity of TCR repertoires was associated with age and clinical stage of the patients with breast cancer. The distribution of Vβ and Jβ genes in TCR repertoires was skewed in patients with human epidermal growth factor receptor 2–positive (HER2+) breast cancer. Vβ20.1 and Vβ30 expression levels before NAC correlate with tumor response after all cycles of NAC in HER2– and HER2+ patients, respectively. Some CDR3 motifs that correlated with clinical response in either HER2+ or HER2– patients were identified. Besides, TCR repertoire evolved during NAC and the diversity of TCR repertoire decreased more after two cycles of NAC in patients with good tumor response after all cycles of NAC ( P = .0061). CONCLUSION Our results demonstrated that TCR repertoire correlated with the characteristics of the tumor, such as the expression status of HER2. Moreover, some characteristics of TCR repertoires that correlated with clinical response were identified and they might provide useful information to tailor therapeutic regimens at the early cycle of NAC.

Reconstitution of T cell immunity against EBV in the immunocompromised host by adoptive transfer of peptide-stimulated T cells after allogeneic stem cell transplantation

Reconstitution of T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by peptide stimulation and adoptively transferred them to a patient with angioimmunoblastic T-cell lymphoma (AITL), who had developed persisting high titers of EBV concomitant to relapse after transplantation. T cell receptor beta (TCRβ) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse.

Donor T-Cell Repertoire Profiling in Recipient Lymphoid and Parenchyma Organs Reveals GVHD Pathogenesis at Clonal Levels After Bone Marrow Transplantation in Mice

The diversity and composition of T-cell receptor (TCR) repertoire, which is the result of V, (D), and J gene recombination in TCR gene locus, has been found to be implicated in T-cell responses in autoimmunity, cancer, and organ transplantation. The correlation of T-cell repertoire with the pathogenesis of graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation remains largely undefined. Here, by utilizing high-throughput sequencing of the genes encoding TCRβ-chain, we comprehensively analyzed the profile of T-cell repertoire in recipient lymphoid and GVHD target organs after bone marrow transplantation (BMT) in mice. In lymphoid organs, TCR diversity was narrowed, accompanied with reduced numbers of unique clones while increased accumulation of dominant clones in allogeneic T cells compared to syngeneic T cells. In an individual allogeneic recipient, donor-derived TCR clones were highly overlapped among tissue sites, and the degree of overlapping was increasing from day 7 to 14 after allogeneic BMT. The top clones in peripheral blood, gut, liver, and lungs were highly mutually shared in an allogenic recipient, indicating that blood has the potential to predict dominant clones in these GVHD target organs. T cells in GVHD target organs from allogeneic recipients had fewer overlapped clones with pre-transplant donor T cells compared to those from syngeneic recipients. Importantly, the top 10 clones in allogeneic recipients were not detectable in pre-transplant donor T cells, indicating clonal expansion of rare rearrangements. Interestingly, even starting from the same pool of donor repertoires, T cells had very few overlapped clones between each allogeneic recipient who developed completely different dominant clones. We were only able to trace a single clone shared by three replicate allogeneic recipients within the top 500 clones. Although dominant clones were different among allogeneic recipients, V26 genes were consistently used more frequently by TCR clones in allogeneic than syngeneic recipients. This is the first study to extensively examine the feature of T-cell repertoire in multiple lymphoid and parenchyma organs, which establishes the association between T-cell activation and GVHD pathogenesis at the level of TCR clones. Immune repertoire sequencing-based methods may represent a novel personalized strategy to guide diagnosis and therapy in GVHD.

T Cell Repertoire Abnormality in Immunodeficiency Patients with DNA Repair and Methylation Defects

Both DNA damage response and methylation play a crucial role in antigen receptor recombination by creating a diverse repertoire in developing lymphocytes, but how their defects relate to T cell repertoire and phenotypic heterogeneity of immunodeficiency remains obscure. We studied the TCR repertoire in patients with the mutation in different genes (ATM, DNMT3B, ZBTB24, RAG1, DCLRE1C, and JAK3) and uncovered distinct characteristics of repertoire diversity. We propose that early aberrancies in thymus T cell development predispose to the heterogeneous phenotypes of the immunodeficiency spectrum. Shorter CDR3 lengths in ATM-deficient patients, resulting from a decreased number of nucleotide insertions during VDJ recombination in the pre-selected TCR repertoire, as well as the increment of CDR3 tyrosine residues, lead to the enrichment of pathology-associated TCRs, which may contribute to the phenotypes of ATM deficiency. Furthermore, patients with DNMT3B and ZBTB24 mutations who exhibit discrepant phenotypes present longer CDR3 lengths and reduced number of known pathology-associated TCRs.

Simultaneous Disruption of XPO1 and A20 in Murine B Cells Influences Both B and T Cell Repertoire

Introduction & Objectives: Efforts to characterize the heterogeneity of advanced hematologic malignancies using large-scale genomic studies have identified recurrent monoallelic mutations affecting the E571 residue of the essential nuclear exporter, Exportin-1 (XPO1; E571K in ~80% of cases, E571G in ~15% of cases). E571-XPO1 mutations alter the charge and structural basis of the cargo-binding region, disrupting critical biophysical interactions between XPO1 and its' cargos. Enriched in hematologic malignancies, E571-XPO1 mutations are predominantly reported in chronic lymphocytic leukemia (CLL; 5-10% of cases), classical Hodgkin's lymphoma (~25% of cases), and primary mediastinal B cell lymphoma (PMBCL; 25-30% of cases). The subsequent change in XPO1-cargo localization alters the transcriptional profile and overall phenotype of the leukemic cell, with evidence suggesting hyper-active NF-κB and NFAT signaling pathways as leading leukemogenic mechanisms. Moreover, while overall immune dysfunction in CLL leads to infections as a major cause of morbidity and mortality, CLL patients with E571-XPO1 mutations are more susceptible to death by infection, suggesting these mutations may exacerbate the leukemia-induced immunosuppressive phenotype. Similarly, inactivating mutations/deletions to A20 (TNFAIP3 gene), the master regulator of NF-κB, are recurrently reported in several B cell malignancies but most frequently observed in PMBCL (~30% of cases). E571-XPO1 mutations and TNFAIP3 deletions/mutations have been found as co-occurring genetic abnormalities in PMBCL, and while TNFAIP3 mutations in CLL are rare, functional convergence on NF-κB and immune signaling suggests altered XPO1 and A20 activity may have unreported pathogenic significance in CLL. Thus, we aimed to explore the oncogenic and immunologic consequence of co-occurring XPO1 and A20 abnormalities by evaluating a novel in-vivo model recapitulating this scenario. Methods: To explore concurrent aberrations to XPO1 and A20, we developed a novel mouse model to recapitulate this event (Eµ-XPO1xA20 KO). This model was generated by crossing the Eµ-XPO1 transgenic mouse - which overexpresses wildtype (WT), E571K, or E571G-XPO1 under control of a VH promoter-IgH-Eµ enhancer to target transgene expression to immature and mature B cells - with a B cell-specific A20 inactivation mouse (A20 KO) - which lacks functional A20 as a result of Cre recombinase-mediated excision of TNFAIP3 exon 3 via loxP recombination sites flanking this region and Cre recombinase expressed under CD19 promoter/enhancer elements. Eµ-XPO1 and Eµ-XPO1xA20 KO mice were aged and followed, and their B and T cell repertoire was assessed via flow cytometry. Results & Conclusion: We previously demonstrated Eµ-XPO1 mice develop a CLL-like disease (CD19+/CD5+/B220dim B lymphocytes), but leukemia development is significantly delayed - evident between 20-30 months of age. Preliminary analysis in adolescent animals revealed irregular lymphocyte populations as early as 6 months of age in the blood and spleen of Eµ-XPO1xA20 KO mice when compared to non-transgenic and Eµ-XPO1 mice; highlighted by elevated populations of CD93+/CD23+ transitional B cells and CD3+ T cells, and reduced populations of CD21+/IgM+ marginal zone B cells. Moreover, development of a circulating CLL-like disease accompanied by palpable lymphadenopathy and splenomegaly was observed in Eµ-XPO1xA20 KO mice as early as 17-20 months of age, again presenting a distinct immunophenotype inconsistent with that observed in Eµ-XPO1 mice. Additionally, progressive accumulation of CD3+/CD19- T cell leukemia-like populations were observed in a subset of Eµ-XPO1xA20 KO and A20 KO mice, indicating these aberrations may further disrupt and stimulate uncontrolled proliferation affecting the overall immune repertoire. Significance: We report that simultaneous disruption of essential regulators XPO1 and A20 in murine B cells encourages development of irregular B and T cell populations, and can stimulate a progressive CLL-like or T cell leukemia-like expansion. Continued investigation with these models can further our understanding of the relationship between overall immune function and these critical regulatory molecules, and can provide considerable insight to identifying pathways for selective targeting as a personalized therapy in several high-risk cancer types. Disclosures Byrd: AstraZeneca: Consultancy; Takada: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Syndex: Consultancy; Trillium: Consultancy; Vincera Pharmaceuticals: Current equity holder in publicly-traded company.

Self-mediated positive selection of T cells sets an obstacle to the recognition of nonself

Adaptive immune recognition is mediated by the binding of peptide–human leukocyte antigen complexes by T cells. Positive selection of T cells in the thymus is a fundamental step in the generation of a responding T cell repertoire: only those T cells survive that recognize human peptides presented on the surface of cortical thymic epithelial cells. We propose that while this step is essential for optimal immune function, the process results in a defective T cell repertoire because it is mediated by self-peptides. To test our hypothesis, we focused on amino acid motifs of peptides in contact with T cell receptors. We found that motifs rarely or not found in the human proteome are unlikely to be recognized by the immune system just like the ones that are not expressed in cortical thymic epithelial cells or not presented on their surface. Peptides carrying such motifs were especially dissimilar to human proteins. Importantly, we present our main findings on two independent T cell activation datasets and directly demonstrate the absence of naïve T cells in the repertoire of healthy individuals. We also show that T cell cross-reactivity is unable to compensate for the absence of positively selected T cells. Additionally, we show that the proposed mechanism could influence the risk for different infectious diseases. In sum, our results suggest a side effect of T cell positive selection, which could explain the nonresponsiveness to many nonself peptides and could improve the understanding of adaptive immune recognition.

Immunogenomic intertumor heterogeneity across primary and metastatic sites in a patient with lung adenocarcinoma

Background: Lung cancer is the leading cause of cancer death, partially owing to its extensive heterogeneity. The analysis of intertumor heterogeneity has been limited by an inability to concurrently obtain tissue from synchronous metastases unaltered by multiple prior lines of therapy. Methods: In order to study the relationship between genomic, epigenomic and T cell repertoire heterogeneity in a rare autopsy case from a young female never-smoker with late-stage lung adenocarcinoma (LUAD), we did whole-exome sequencing (WES), DNA methylation and T-cell receptor (TCR) sequencing to characterize the immunogenomic landscape of one primary and 19 synchronous metastatic tumors. Results: We observed heterogeneous mutation, methylation, and T cell patterns across distinct metastases including a set of prevalent T cell clonotypes which were completely excluded from left-side thoracic tumors. Though a limited number of predicted neoantigens were shared, these were associated with homology of the T cell repertoire across metastases. Lastly, ratio of methylated neoantigen coding mutations was negatively associated with T-cell density, richness and clonality, suggesting neoantigen methylation may partially drive immunosuppression. Conclusions: Our study demonstrates heterogeneous genomic and T cell profiles across synchronous metastases and how restriction of unique T cell clonotypes within an individual may differentially shape the genomic and epigenomic landscapes of synchronous lung metastases.