Low T-cell Receptor Research Articles

A GM-CSF-neuroantigen tolerogenic vaccine elicits inefficient antigen recognition events below the CD40L triggering threshold to expand CD4+ CD25+ FOXP3+ Tregs that inhibit experimental autoimmune encephalomyelitis (EAE)

BackgroundTolerogenic vaccines represent antigen-specific interventions designed to re-establish self-tolerance and thereby alleviate autoimmune diseases, which collectively comprise over 100 chronic inflammatory diseases afflicting more than 20 million Americans. Tolerogenic vaccines comprised of single-chain GM-CSF-neuroantigen (GMCSF-NAg) fusion proteins were shown in previous studies to prevent and reverse disease in multiple rodent models of experimental autoimmune encephalomyelitis (EAE) by a mechanism contingent upon the function of CD4+ CD25+ FOXP3+ regulatory T cells (Tregs). GMCSF-NAg vaccines inhibited EAE in both quiescent and inflammatory environments in association with low-efficiency T cell receptor (TCR) signaling events that elicited clonal expansion of immunosuppressive Tregs.MethodsThis study focused on two vaccines, including GMCSF-MOG (myelin oligodendrocyte glycoprotein 35–55/MOG35–55) and GMCSF-NFM (neurofilament medium peptide 13–37/NFM13–37), that engaged the transgenic 2D2 TCR with either low or high efficiencies, respectively. 2D2 mice were crossed with FOXP3 IRES eGFP (FIG) mice to track Tregs and further crossed with Rag−/− mice to reduce pre-existing Treg populations.ResultsThis study provided evidence that low and high efficiency TCR interactions were integrated via CD40L expression levels to control the Treg/Tcon balance. The high-efficiency GMCSF-NFM vaccine elicited memory Tcon responses in association with activation of the CD40L costimulatory system. Conversely, the low-efficiency GMCSF-MOG vaccine lacked adequate TCR signal strength to elicit CD40L expression and instead elicited Tregs by a mechanism that was impaired by a CD40 agonist. When combined, the low- and high-efficiency GMCSF-NAg vaccines resulted in a balanced outcome and elicited both Tregs and Tcon responses without the predominance of a dominant immunogenic Tcon response. Aside from Treg expansion in 2D2-FIG mice, GMCSF-MOG caused a sustained decrease in TCR-β, CD3, and CD62L expression and a sustained increase in CD44 expression in Tcon subsets. Subcutaneous administration of GMCSF-MOG without adjuvants inhibited EAE in wildtype mice, which had a replete Treg repertoire, but was pathogenic rather than tolerogenic in 2D2-FIG-Rag1−/− mice, which lacked pre-existing Tregs.ConclusionsThis study provided evidence that the GMCSF-MOG vaccine elicited antigenic responses beneath the CD40L triggering threshold, which defined an antigenic niche that drove dominant expansion of tolerogenic myelin-specific Tregs that inhibited EAE.

EuroFlow Standardized Approach to Diagnostic Immunopheneotyping of Severe PID in Newborns and Young Children.

The EuroFlow PID consortium developed a set of flow cytometry tests for evaluation of patients with suspicion of primary immunodeficiency (PID). In this technical report we evaluate the performance of the SCID-RTE tube that explores the presence of recent thymic emigrants (RTE) together with T-cell activation status and maturation stages and discuss its applicability in the context of the broader EuroFlow PID flow cytometry testing algorithm for diagnostic orientation of PID of the lymphoid system. We have analyzed peripheral blood cells of 26 patients diagnosed between birth and 2 years of age with a genetically defined primary immunodeficiency disorder: 15 severe combined immunodeficiency (SCID) patients had disease-causing mutations in RAG1 or RAG2 (n = 4, two of them presented with Omenn syndrome), IL2RG (n = 4, one of them with confirmed maternal engraftment), NHEJ1 (n = 1), CD3E (n = 1), ADA (n = 1), JAK3 (n = 3, two of them with maternal engraftment) and DCLRE1C (n = 1) and 11 other PID patients had diverse molecular defects [ZAP70 (n = 1), WAS (n = 2), PNP (n = 1), FOXP3 (n = 1), del22q11.2 (DiGeorge n = 4), CDC42 (n = 1) and FAS (n = 1)]. In addition, 44 healthy controls in the same age group were analyzed using the SCID-RTE tube in four EuroFlow laboratories using a standardized 8-color approach. RTE were defined as CD62L+CD45RO-HLA-DR-CD31+ and the activation status was assessed by the expression of HLA-DR+. Naïve CD8+ T-lymphocytes and naïve CD4+ T-lymphocytes were defined as CD62L+CD45RO-HLA-DR-. With the SCID-RTE tube, we identified patients with PID by low levels or absence of RTE in comparison to controls as well as low levels of naïve CD4+ and naïve CD8+ lymphocytes. These parameters yielded 100% sensitivity for SCID. All SCID patients had absence of RTE, including the patients with confirmed maternal engraftment or oligoclonally expanded T-cells characteristic for Omenn syndrome. Another dominant finding was the increased numbers of activated CD4+HLA-DR+ and CD8+HLA-DR+ lymphocytes. Therefore, the EuroFlow SCID-RTE tube together with the previously published PIDOT tube form a sensitive and complete cytometric diagnostic test suitable for patients suspected of severe PID (SCID or CID) as well as for children identified via newborn screening programs for SCID with low or absent T-cell receptor excision circles (TRECs).

Immuno-genomic landscape of osteosarcoma

Limited clinical activity has been seen in osteosarcoma (OS) patients treated with immune checkpoint inhibitors (ICI). To gain insights into the immunogenic potential of these tumors, we conducted whole genome, RNA, and T-cell receptor sequencing, immunohistochemistry and reverse phase protein array profiling (RPPA) on OS specimens from 48 pediatric and adult patients with primary, relapsed, and metastatic OS. Median immune infiltrate level was lower than in other tumor types where ICI are effective, with concomitant low T-cell receptor clonalities. Neoantigen expression in OS was lacking and significantly associated with high levels of nonsense-mediated decay (NMD). Samples with low immune infiltrate had higher number of deleted genes while those with high immune infiltrate expressed higher levels of adaptive resistance pathways. PARP2 expression levels were significantly negatively associated with the immune infiltrate. Together, these data reveal multiple immunosuppressive features of OS and suggest immunotherapeutic opportunities in OS patients.

Low T-Cell Receptor β ( ) Repertoire Diversity Early Post-Transplant for Severe Combined Immunodeficiency (SCID) Predicts Subsequent Failure of Immune Reconstitution

Introduction Development of a diverse T cell repertoire is associated with immune recovery following allogeneic hematopoietic cell transplantation (HCT) for SCID. Although CDR3 spectratyping and staining of Vβ families provide approximations of the diversity of T cell receptors, high-throughput sequencing (HTS) of the TRB repertoire allows precise evaluation of clonotype dynamics during immune reconstitution. We investigated whether longitudinal HTS analysis of TRB would accurately assess development of TCR repertoire diversity over time and reflect the quality of T cell reconstitution following HCT for SCID. We also studied the influence of SCID genotype, conditioning regimen, donor type, and occurrence of chronic graft versus host disease (cGVHD) on TCR diversity post HCT. We hypothesized that repertoire diversity may represent an early biomarker to predict long-term immune reconstitution vs. need for a second intervention. Methods HTS was used to study the composition and diversity of TRB repertoire of 27 SCID infants, pre-HCT and at 100 d, 6 and 12 mo and yearly post-treatment(s). Median time of follow-up was 48 mo. Subjects were part of a prospective study of SCID by the Primary Immunodeficiency Treatment Consortium. Equal amounts of total RNA extracted from peripheral blood was used as template to amplify TRB rearrangements. The VDJ statistics file (PAST program) was used to calculate a Shannon entropy (H) index to measure repertoire diversity. Results TRB sequence analysis of 27 SCID patients showed poor diversity at baseline, followed by improvement to normal complexity (H index >8.0) after HCT. Similar kinetics of development of TRB diversity were seen in patients with IL2RG, JAK3, and IL7R defects (n=16) as in those with RAG and Artemis defects (n=14). In the latter group, however, HCT with no conditioning or immune suppression only was associated with persistently lower diversity than HCT with conditioning (p 500 CD4+ T cells/ul or > 200 naive CD4+ T cells/ul at 6 mo post-HCT correlated with higher TRB diversity at 24 and 36 mo post-HCT (p Conclusions Analysis of TRB diversity permits a detailed assessment of development of a diverse T cell repertoire following cellular therapies for SCID and confirms the need for patient-tailored treatment strategies based on SCID genotype. We propose that analysis of TRB diversity at 100 d post-HCT may identify patients at risk for failure of sustained immune reconstitution, thus prompting a second intervention without delay.

T cell receptor repertoire as a prognosis marker for heat shock protein peptide complex-96 vaccine trial against newly diagnosed glioblastoma

ABSTRACT Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults with a dismal prognosis. We previously reported that vaccination with heat shock protein peptide complex-96 (HSPPC-96) improves survival in patients with newly diagnosed GBM (NCT02122822). Especially for patients with a strong antitumor immune response after vaccination, a durable survival benefit can be achieved. Here, we conducted T cell receptor (TCR) sequencing to retrospectively examine the TCR repertoires of tumor-infiltrating lymphocytes in long-term survivors (LTS) and short-term survivors (STS). We found that LTS exhibit lower TCR repertoire diversity compared with STS, indicating the prevalence of dominant TCR clones in LTS tumors. Accordingly, the LTS group showed increased inter-patient similarity, especially among high-frequency TCR clones, implying some of these dominant clones are shared among LTS. Indeed, we discovered four TCR clones significantly enriched in the LTS group: the presence of these clones has predictive value for stratifying patients prior to vaccination. Together, these findings uncover a group of preexisting TCR clones shared in LTS that can be utilized as candidate biomarkers to select GBM patients most likely to durably respond to HSPPC-96 treatment.

High Frequency Production of T Cell-Derived iPSC Clones Capable of Generating Potent Cytotoxic T Cells

Current adoptive T cell therapies conducted in an autologous setting are costly, time-consuming, and depend on the quality of the patient’s T cells, and thus it would be highly beneficial to develop an allogeneic strategy. To this aim, we have developed a method by which cytotoxic T lymphocytes (CTLs) are regenerated from induced pluripotent stem cells that are originally derived from T cells (T-iPSCs). In order to assess the feasibility of this strategy, we investigated the frequency of usable T-iPSC clones in terms of their T cell-generating capability and T cell receptor (TCR) affinity. We first established eight clones of T-iPSCs bearing different MART-1-specific TCRs from a healthy volunteer. Whereas all clones were able to give rise to mature CTLs, cell yield varied greatly, and five clones were considered to be usable. TCR affinity in the regenerated CTLs showed a large variance among the eight clones, but functional avidities measured by cytotoxic activity were almost equivalent among three selected clones representing high, medium, and low TCR affinity. In a total of 50 alloreactivity tests using five CTL clones versus ten target cells, alloreactivity was seen in only three cases. These findings collectively support the feasibility of this T-iPSC strategy.

Detecting 22q11.2 Deletion Syndrome in Newborns With Low T Cell Receptor Excision Circles From Severe Combined Immunodeficiency Screening

HC Liao, CH Liao, SM Kao, CC Chiang, YJ Chen. J Pediatr. 2019;204:219–224.e1 To evaluate the prevalence of 22q11.2 deletion syndrome within a set of newborns screening positive for low T-cell receptor excision circles (TRECs) by assessing a patient’s copy number of the TBX1 and/or HIRA genes, which are located in the 22q11.2 region. This population evaluated 486 newborns born in Taiwan who were diagnosed with low TRECs (<90 copies per microliter) on the severe combined immunodeficiency newborn screening …

Comprehensive immune characterization and T-cell receptor repertoire heterogeneity of retroperitoneal liposarcoma.

Retroperitoneal liposarcoma (RLPS) is one of the most common subtypes of retroperitoneal soft tissue sarcomas and lacks effective treatment. This study aimed to provide a thorough profile of immune characteristics of RLPS. This study included 56 RLPS patients. Multisite tumor tissues were collected from 16 patients. Immunohistochemistry was carried out to identify CD4+, CD8+, FoxP3+, CD20+, or programmed cell death‐1 (PD‐1)+ tumor infiltrating lymphocytes (TILs) and Programmed cell death ligand‐1 (PD‐L1) expression in tumor tissues. Ultradeep sequencing of T‐cell receptor (TCR) β‐chain gene was carried out in 42 tumor samples as well as peripheral blood samples collected from 6 patients. In RLPS, TILs were distributed in 3 patterns and T cells were more prevalent than B cells. Generally, the proportion of TILs decreased and PD‐L1 expression increased with tumor progression. Patients with higher PD‐1/PD‐L1 expression tended to have poorer prognosis, whereas patients with tertiary lymphoid structure tended to have a favorable disease‐free survival. Although T‐cell clones in tumors were quite different from those in peripheral blood, TCR sequencing showed low TCR repertoire reads as well as polyclonal status within tumors, which indicated limited T cell response in the tumors. Both TILs distribution and TCR repertoires suggested spatial immune heterogeneity in RLPS. Our research described the immune landscape of RLPS, and suggested RLPS might be a kind of tumor with low T cell infiltration as well as great immune heterogeneity. Therefore, strategies that can facilitate lymphocytic infiltration and immune reactivity need to be developed in the future to improve the efficacy of immunotherapy.

Measuring the T-cell down-regulation of TCR-zeta, ZAP-70 and CD28 in arthritis patients: An old tool for new biomarkers.

Low T-cell receptor (TCR)/CD28 signaling lymphocytes are expanded in arthritis. We asked whether the down-expression of TCR-related molecules correlates with specific arthritis characteristics and if it has clinical implications. TCR-ZETA, ZAP-70 and CD28 expression was measured by flow cytometry in synovial fluid (SF) and peripheral blood (PB)-derived T cells. In PB, ZETA-downregulation in CD4+ CD28+ and consequent CD4+ CD28lowZETAlow cell expansion correlate with CRP elevation, leukocyte recruitment into SF and, primarily, disease activity (DAS). In some patients, ZETA-downregulation extends to CD8+ CD28null and/or CD8+ CD28+ cells, and this correlates with enhanced leukocyte recruitment, multiple joint involvement, and disability index (HAQ). ZETA-downregulation in CD4+ CD28+ may also lead to CD4+ CD28+ ZETAnull cell expansion, which strongly correlates with HAQ. In SF, ZETA-downregulation in CD8+ CD28null and consequent CD8+ CD28nullZETAlow/null cell expansion correlate with CRP elevation and neutrophilic influx into SF, whereas ZAP-downregulation in CD8+ CD28+ and consequent CD8+ CD28lowZAPlow cell expansion strongly correlate with HAQ and DAS. ZETA-downregulation is preponderant in SF of seronegative arthritides, with seronegative rheumatoid arthritis showing significant down-regulation in CD8+ CD28null, and non-rheumatoid arthritides showing significant down-regulation in CD4+ CD28+ . Altogether, we identified new molecular and cellular biomarkers of arthritis-related T-cell inflammation, useful for assessing arthritis activity, predicting polyarticular progression and functional impairment, characterizing seronegative arthritides, and possibly tailoring immunotherapies.

Abstract 2238: Characterization of T cell clonality and tumor mutation burden utilizing the TCRSeq NGS and the comprehensive NGS assays

Abstract Background: Somatic mutations are the basis for the generation of potential neoantigens recognized by antitumor T lymphocytes, which may contribute to increase T cell diversity [1]. Next-generation sequencing of expressed T cell receptor (TCR) transcripts allows for quantification and assessing clonality of T cell diversity [2]. Tumors that exhibit a low TCR diversity and potentially having a high clonality have been shown to be lower responders to immunotherapy treatments. Methods: Utilizing the Shannon entropy to arrive at a clonality readout, we sought to gain mechanistic insights and demonstrate a link between clonality and total mutation load. We conducted the analysis using a diverse set of solid tumors and hematological malignancies across three different specimen types (bone marrow (BM), peripheral blood (PB), formalin-fixed paraffin embedded (FFPE tissue)). Specifically, we used the beta TCRSeq NGS and the Comprehensive NGS assays (internally developed) to characterize sample clonality and mutation load respectively. Results: The TCRSeq NGS assay sequences the V(D)J domains of the variable region of the TCR heavy chain (involved in epitope recognition), while the hybrid capture Comprehensive NGS assay detects variants across a 236 gene panel optimized for SNV and INDEL detection. Initial analysis across the different specimen types resulted in average clonalities of BM = 0.255, PB = 0.149, FFPE = 0.092, with average tumor mutation loads of 7.6, 7.6, and 21 respectively. Categorization of all samples by increasing clonality, independent of specimen type, established a clear differentiator with samples harboring a clonality of less than 0.10 to exhibit an average mutation load of 26.33. This was statistically significant from samples harboring clonalities between 0.10 and 0.19, which had an average mutation load of 7.86, and samples with clonalities equal to or greater than 0.20, having an average mutation load of 7.67. Conclusions: Based on two different categorical analyses, lower T cell clonality readouts appear to be associated with solid tumors harboring greater genetic diversity/neoantigen burden than hematological malignancies with higher clonalities. As tumor mutation burden has been correlated with clinical outcomes of varying efficacy, this study provides further insight into the utility of T cell clonality as a “molecular tag” for immune response and resistance in oncological therapeutics. Citation Format: Hua C. Gong, Hua Gong, Andreas Papoutsis, Wen Wu, Christian Laing. Characterization of T cell clonality and tumor mutation burden utilizing the TCRSeq NGS and the comprehensive NGS assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2238.

Abstract 1091: Comparison of TCR repertoires between cancer tissues and lymph nodes in colorectal cancer patients

Abstract Tumor draining lymph nodes (TDLNs) are located in the routes of lymphatic drainage from a primary tumor and have the highest metastasis in various types of solid tumors. TDLNs are considered as a tissue to activate the antitumor immunity, where antigen-specific effector T cells are generated and released to the blood stream. These effector T cells then infiltrate into the tumor site and attack cancer cells. However, T cell receptor (TCR) repertoire in TDLNs has not been well characterized. We performed TCR sequencing of cancer tissues, corresponding normal mucosa tissues and a total of 203 regional lymph nodes, including 67 metastasis-positive lymph nodes from 23 colorectal cancer patients with lymph node metastasis. Metastasis-positive lymph nodes showed a significantly lower TCR diversity and shared TCR clones more frequently with primary tumor tissues compared to metastasis-negative TDLNs. Hierarchical clustering and principal component analyses also supported that TCR repertoires in metastasis-positive TDLNs were more similar to primary tumor tissues than metastasis-negative TDLNs. These finding suggest that cancer-reactive T cell clones might expand in the metastasis-positive TDLNs, although we have not tested their reactivity against cancer cells. We are now examining whether the T cells in TDLNs can recognized autologous cancer cells. Citation Format: Kazuma Kiyotani, Tatsuo Matsuda, Eisaku Miyauchi, Yu-Wen Hsu, Satoshi Nagayama, Makda Zewde, Jae-Hyun Park, Taigo Kato, Makiko Harada, Nobuaki Suzuki, Hiroaki Nagano, Shoichi Hazama, Yusuke Nakamura. Comparison of TCR repertoires between cancer tissues and lymph nodes in colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1091.

TCR sequencing analysis of cancer tissues and tumor draining lymph nodes in colorectal cancer patients

ABSTRACTTumor draining lymph nodes (TDLNs) are located in the routes of lymphatic drainage from a primary tumor and have the highest risk of metastasis in various types of solid tumors. TDLNs are also considered as a tissue to activate the antitumor immunity, where antigen-specific effector T cells are generated. However, T cell receptor (TCR) repertoires in TDLNs have not been well characterized. We collected 23 colorectal cancer tumors with 203 lymph nodes with/without metastatic cancer cells (67 were metastasis-positive and the remaining 136 were metastasis-negative) and performed TCR sequencing. Metastasis-positive TDLNs showed a significantly lower TCR diversity and shared TCR clonotypes more frequently with primary tumor tissues compared to metastasis-negative TDLNs. Principal component analysis indicated that TDLNs with metastasis showed similar TCR repertoires. These findings suggest that cancer-reactive T cell clones could be enriched in the metastasis-positive TDLNs.

Focused Regulatory T Cell Repertoires Are Indicative of Successful GvHD Control in Experimental and Clinical Stem Cell Transplantation

Acute Graft versus Host Disease (aGvHD) remains a major complication and leading cause of mortality after allogeneic stem cell or bone marrow transplantation (BMT). Current strategies for treatment are still based on unspecific immunosuppressive therapy. Over the last decade, there have been major advances in the field of adoptive immunotherapy using regulatory CD4+CD25+Foxp3+ T cells (Treg cells). Nonetheless, not much is known about the exact mechanisms of Treg-mediated suppression, and even less about the importance of T cell receptor (TCR) specificity and its diversity on the functionality of Tregs.We hypothesized that an optimal Treg TCR repertoire is necessary for successful prevention of aGvHD. To test this hypothesis, we sequenced the TCR repertoire of 8 patients who were diagnosed with aGvHD on day 30 post transplantation and compared it with the TCR repertoire of nine GvHD-free patients. Analysis of GvHD-free patients on day 30 (and 100 days-follow up) revealed a lower TCR diversity when compared to the patients suffering from GvHD. A more detailed analysis of the TCR repertoire showed that in patients without GvHD, fewer clonotypes were needed to comprise 50% of the whole repertoire as compared with samples from patients with GvHD (Figure 1A). Thus, expansion of protective clones indicates their potent immunosuppressive capabilities.Next, we employed a well-described murine model of allogeneic BMT (BL/6–>Balb/c) with co-injection of Tregs. Recipient Balb/c mice transplanted in this fashion were previously shown to be protected from aGvHD. However, the mechanisms involved in this Treg-mediated protection are not fully understood. Therefore, Tregs were FACS sorted from B6.Cg-Foxp3tm1Mal/J mice based on their Foxp3 expression. Recipient mice were transplanted with T-cell depleted bone marrow and a mixture of conventional T cells (Tconv) and Tregs in 1:1 ratio. Transferred Tregs were re-sorted on day 7 and day 14 from secondary lymphoid organs based on the congenic marker Thy 1.1 and Foxp3 expression. Using this model, we investigated the kinetics of the Treg TCR repertoire early after BMT in 5 independent experiments. We found a consistently similar narrowing of the repertoire and clonal expansion in mice protected from GvHD (Figure 1B). Diversity analysis using inverse Simpson Index also confirmed our findings. These data further support the notion that a clonal expansion of Tregs is necessary for an optimal immunosuppression of an allogeneic response, both in human and in mice. To test the functionality and phenotype of such expanded Tregs, they were re-sorted from BMT-recipient mice 14 days after transplantation. These Tregs were expanded using α-CD3 and α-CD28 antibodies and were functionality tested in an in vitro Treg suppression assay. Re-sorted Tregs after expansion showed expression of established Treg surface and intracellular markers such as Helios, CD25, GITR and CTLA-4. For the suppression assay, responder CD4 Tconv were stained with a proliferation tracking dye eFluor670 and stimulated in vitro with CD3 and CD28 beads in the presence of different ratios of re-sorted and expanded, or polyclonaly activated Tregs as the control. Allo-specific ex vivo Tregs exhibited a superior suppressive potential when compared with polyclonaly activated Tregs in vitro.Taken together, our current study highlights the importance of specific Treg driven allo-response in GvHD prevention. Further studies are needed, particularly in larger patient cohorts to confirm these findings. However, we propose that this approach might lead to identification and subsequent use of specific Treg clones with high immunosuppressive capacity for the prevention of aGvHD. [Display omitted] DisclosuresGanser:Novartis: Membership on an entity's Board of Directors or advisory committees. Koenecke:abbvie: Consultancy; BMS: Consultancy; Roche: Consultancy; Amgen: Consultancy.

The Diminished Neoantigens with Copy Number Aberrations and Diverse TCR Repertoire Shorten the Recurrence-Free Survival Period in Colorectal Cancer Cases

Background: We previously disclosed the evolutionary principle shaping intratumor heterogeneity shifts from Darwinian to neutral evolution in primary colorectal cancer (CRC) progression. Here, we focused on the role of copy number aberrations (CNAs) in impairment of the host tumor immune response, which is characterized by the level of neoantigen (NAG) and diversity of the T-cell receptor (TCR) repertoire, and in fostering postoperative recurrence. Methods: We compared single nucleotide variants (SNVs), NAG, and CNAs between primary tumors from eight cases of early CRC with postoperative recurrence (CRCR) and from eight cases of precancerous CRC lesions without recurrence (PCRC) by whole-exome sequencing. Then, we compared tumor immune response-related factors, such as NAG, cytolytic activity (CYT), and TCR repertoire diversity, between eight primary and 12 metastatic sites from eight CRCR cases. Findings: In comparison of 10 primary CRC tumors with recurrence (CRCR) and eight precancerous tumors of CRC (PCRC), neoantigens were fewer in CRCR than in PCRC, and CNAs at the arm level were significantly more frequent in CRCR than in PCRC. SNVs and copy numbers (CNs) were inversely associated. In TCGA analysis, cytotoxic T lymphocyte-related genes CD8A, CD4G, and PD-1 in 48 primary tumors with recurrence were downregulated relative to those in 242 tumors without recurrence. In CRCR cases, the frequency of CNAs and mutated driver genes with clonality in primary sites were equal to those in recurrent sites; however, the recurrence-free period was significantly extended under low NAG level and diverse TCR repertoire in metastatic but not primary sites. Interpretation: During the evolution of CRC toward recurrence, CNAs determine the metastatic capability with diminished levels of NAG in primary tumors, and impaired NAG and diminished TCR repertoire diversity predominantly determine the recurrence-free survival period. Funding Statement: This project was supported by Japan Society for the Promotion of Science KAKENHI (16K19107, 15H05707), Grant-in-Aid for Scientific Research on Innovative Areas (15H0912), and Priority Issue on Post-K computer (hp170227, hp160219) and partially supported by the Project for Cancer Research and Therapeutic Evolution (P-CREATE) and Practical Research for Innovative Cancer Control from Japan Agency for Medical Research and Development, AMED. The Takamatsunomiya Cancer Research Funding. This research used the supercomputing resource provided by the Human Genome Center, Institute of Medical Science, University of Tokyo. Declaration of Interests: There are no conflicts of interest to declare. Ethics Approval Statement: The study design was approved by the institutional review boards and ethics committees of the hospitals to which the patients were admitted (Kyushu University Hospital Institutional Review Board: protocol number 486–01; Cancer Institute Hospital Institutional Review Board: protocol number 12–27). The study was conducted according to the principles expressed in the Declaration of Helsinki. We obtained written informed consent from all participants in this study.

Abstract 1005: Combination T cell receptor immunosequencing and multiplex immunohistochemistry reveal novel insights into the immune response to human pancreatic cancer

Abstract Background: Despite advancements in therapy, pancreatic ductal adenocarcinoma (PDA) remains an aggressive cancer with high mortality. It is characterized by dense inflammation, including many T cells; however, it is unclear whether these T cells signify true anti-tumor response. In the setting of disappointing results of immunotherapy in PDA, we sought to gain a deeper understanding of the intratumoral T cell response. Methods: With IRB approval, we obtained archival resected PDA tumors in paraffin embedded blocks from 54 patients and performed T cell receptor (TCR) immunosequencing on extracted DNA. Productive clonality was defined as 1-Pielou's evenness; TCR fraction was calculated from number of observed templates versus housekeeping genes. Multiplex immunohistochemistry (IHC) was performed on slides immediately adjacent to blocks sent for TCR sequencing using the PerkinElmer Vectra system. False H&amp;E images generated from autofluorescence signatures were analyzed by a pathologist to categorize peritumoral stroma versus lymphoid tissue. Image analysis was performed using FlowJo10, turning object fluorescence data into cytometric outputs. Correlation analysis between multiplex and TCR data was performed using Pearson's correlation. Results: Mean TCR fraction was 0.27, and mean clonality was 0.15 (typical peripheral blood clonality is 0.08). TCR fraction was positively correlated with clonality (R2=0.23, p=0.007), but there was no survival difference between high or low TCR fraction or clonality. 3.1 million individual cells within the peritumoral stroma were phenotyped in total using multiplex IHC; within the stroma, CD4 T cells, CD8 T cells, and macrophages made up on average 4.6%, 6.8%, and 2.0% of all cells analyzed. 35.6% of CD8 T cells were positive for PD-1, while 20.8% of CD4 cells were FOXP3+ regulatory T cells (Treg). TCR fraction correlated positively with infiltration of CD8+ (R2=0.28, p=0.03) and CD68+ (R2=0.27, p=0.03) cells, but not CD4 cells. Only CD8 cells which were PD-1- correlated positively with clonality (R2=0.26, p=0.04). The presence of Treg correlated very strongly with CD8+PD-1+ cells (R2=0.44, p=0.0004). Conclusion: Here we demonstrate a novel approach to describe the varying immunophenotypic landscape of human PDA depending on the clonal expansion of intratumoral T cells. T cell infiltration appear to be correlated mostly with CD8 T cells as well as the presence of macrophages. Patients with more clonal expansion also had more CD8 cells that lacked the exhaustion marker PD-1, and the presence of regulatory T cells accompanied environments where CD8 cells were positive for PD-1. These data suggest that cytotoxic T cell exhaustion is a phenomenon which may be regulated by Treg and have a negative impact on clonal expansion of intratumoral T cells in human PDA. Citation Format: Yongwoo David Seo, Florencia Jalikis, Xiuyun Jiang, Kevin M. Sullivan, Marissa Vignali, Harlan Robins, Venu G. Pillarisetty. Combination T cell receptor immunosequencing and multiplex immunohistochemistry reveal novel insights into the immune response to human pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1005.

Abstract 4057: Identification of DNA repair genes that affect clonal heterogeneity and the immune microenvironment in breast cancers

Abstract Background: While genetic aberrations of DNA repair genes are known to increase immunogenicity in breast cancer, the clinical significance of the tumor microenvironment including tumor-infiltrating immune cells in regard to DNA repair genes has not yet been elucidated in breast cancer patients. We sought to determine the significance of DNA repair genes and their impact on the immune microenvironment, which could provide insight for the development of immunotherapy in breast cancer. We aimed to correlate DNA repair gene deficiency with tumor-infiltrating immune cells and tumor immune microenvironment, and assess its clinical relevance. Materials and Methods: Integrated and unbiased transcriptomics approach was conducted on genomic and clinical information of 3,614 breast cancer patients of The cancer Genome Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) to assess the association between the aberration of DNA repair genes and the tumor immune microenvironment in breast cancer, as well as the clinical significance utilizing an integrated immunogenomic pipeline. Tumor-infiltrating immune cells (TICs) composition was calculated using the CIBERSORT system that estimates the fraction of 22 immune cell types in each tumor. Intratumor clonal heterogeneity and T-cell receptor diversity was evaluated utilizing mutant allele tumor heterogeneity (MATH) index and TCR repertoire analysis. Results: We found that low expression of DNA repair genes, especially low BRCA1 expression, was significantly associated with improved survival and higher CD8+ cell composition. Tumors with low expression of BRCA1 enriched immune-response related gene sets, using gene set enrichment analysis, significantly associated to higher expression of the immune gene signature, such as HLA-A, PRF1, GZMA, and GZMB, as well as local immune cytolytic activity (CYT). Intratumor clonal heterogeneity was associated with low expression of BRCA1 with high composition of CD8+ T-cells. T-cell receptor (TCR) repertoire analysis elucidated that lower TCR diversity correlated to low BRCA1 expression in hormone-negative or triple-negative breast cancer patients. Utilizing the GEO database with clinical information of response to neoadjuvant chemotherapy (NAC) in breast cancer, patients with low BRCA1 expression showed significantly better response to NAC. Conclusions: We conclude that the comprehensive and unbiased immunogenomic analysis clearly elucidated that low expression of BRCA1, was associated with higher intratumor immune response including immune gene signature, TICs, intratumor clonal heterogeneity, and TCR diversity, all of which have a significant impact on the clinical outcome in breast cancer. Citation Format: Tstutomu Kawaguchi, Xuan Peng, Qianya Qi, Kerry-Ann McDonald, Jessica Young, Song Liu, Eigo Otsuji, Li Yan, Kazuaki Takabe. Identification of DNA repair genes that affect clonal heterogeneity and the immune microenvironment in breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4057.

Distinct immune signatures in chronic lymphocytic leukemia (CLL) and Richter’s syndrome (RS).

7524 Background: Immune checkpoint blockade with PD-1 inhibitor pembrolizumab was effective in RS but not CLL in a phase 2 study (NCT02332980). Analyzing the immune signatures including PD-L1 expression and T cell diversity is important for understanding the differential responses. Methods: 15 CLL and 14 RS patients in NCT02332980 were included. Expression of PD-L1 in lymph node (LN) was analyzed by immunohistochemistry staining. Peripheral blood (PB) and LN T cell diversity was analyzed using the ImmunoSEQ platform (Adaptive Biotechnology), which quantifies the clonailty of T cells by deep sequencing of the CDR3 region of T cell receptor (TCR). Data analysis (Student’s t-test and Mann-Whiney U test) was done by GraphPad Prism (v7). Results: PD-L1 expression was significantly lower in CLL (n = 6) vs RS (n = 12) patients (mean 5.5% vs 22.9%, P= 0.003). A control CLL cohort (n = 11, Mayo CLL tissue registry) also had lower PD-L1 expression (7.7%, P= 0.002 vs RS). RS Patients progressed on ibrutinib or chemotherapy had similar PD-L1 expression (25.3% vs 19.6%, P= 0.416). PB TCR clonality at trial baseline was significantly lower in RS (n = 13) vs CLL (n = 13) patients (median 0.098 vs 0.342, P= 0.026). Patients progressed on ibrutinib or chemotherapy had similar TCR clonality (CLL: 0.34 vs 0.24, P= 0.524; RS: 0.11 vs 0.09, P= 0.534). 5 RS patients achieved an objective response (1 CR, 2 PR, 1 CMR and 1 PMR), 4 of which were after progression on ibrutinib. For CLL patients, only 1 nodal reduction was seen. 4 RS patients had prior CLL samples available for TCR analysis. 2 patients had a notable decrease of TCR clonality (0.21-0.04 and 0.17-0.03) from CLL (PB) to RS state (LN), and achieved CR and PR, respectively. 2 other patients with stable clonality (0.04-0.05 and 0.03-0.08) had SD and PMR, respectively. PB TCR clonality did not change significantly with pembrolizumab (CLL [n = 11]: P= 0.775; RS [n = 11]: P= 0.830; paired t-test). Conclusions: RS patients had higher expression of PD-L1 and lower TCR clonality (more diverse T cells) compared with CLL patients. The distinct immune signatures may explain their differential responses to PD-1 blockade. The dynamic changes of TCR clonality during Richter’s transformation may predict a response to PD-1 blockade.

TCR deep sequencing of transgenic RAG-1-deficient mice reveals endogenous TCR recombination: a cause for caution.

The utility of T-cell receptor (TCR) transgenic mice in medical research has been considerable, with applications ranging from basic biology all the way to translational and clinical investigations. Crossing of TCR transgenic mice with either recombination-activating gene (RAG)-1 or RAG-2 knockouts is frequently used to generate mice with a monoclonal T-cell repertoire. However, low level productive TCR rearrangement has been reported in RAG-deficient mice expressing transgenic TCRs. Using deep sequencing, we set out to directly examine and quantify the presence of these endogenous TCRs. Our demonstration that functional nontransgenic TCRs are present in nonmanipulated mice has wide reaching ramifications worthy of critical consideration.

Case of 22q11.2 Deletion Syndrome Not Identified by &lt;i&gt;TBX1&lt;/i&gt; Screening with a Positive SCID Newborn Screen

Individuals with 22q11.2 deletion syndrome (22q11.2DS) have an embryological midline fusion defect, which can result in a syndrome including congenital heart disease, cleft palate, hypoparathyroidism, thymic hypoplasia, immunologic abnormalities, and developmental delay. The majority of patients have a 3 megabase deletion, which contains multiple genes, including the T-box transcription factor (TBX1) gene. Definitive diagnosis is made through fluorescent in situ hybridization (FISH) or chromosomal microarray (CMA). Newborn screening (NBS) for severe combined immune deficiency (SCID) via low T-cell receptor excision circles (TRECs) can also identify this population if thymic output is low. Samples from infants who screen positive undergo further testing, which includes a purine profile and TBX1 deletion analysis. Diagnostic follow-up testing is centre-dependent and may not include more definitive testing for 22q11.2DS by FISH or CMA. We report a case of a newborn with 22q11.2DS, detected by low TRECs on the SCID NBS, with a normal screening TBX1 result, who was subsequently found to have a TBX1 gene deletion on follow-up 22q11.2 FISH and CMA. This case highlights the limitations of the TBX1 screening assay and the importance of performing diagnostic testing with FISH and (or) CMA regardless of the initial TBX1 result. It also emphasizes the need for a standardized follow-up testing algorithm across institutions for newborns who screen positive for SCID. Statement of novelty: To our knowledge, this is the first case to be described in the literature where a newborn with 22q11.2DS with a typical deletion encompassing the TBX1 gene was not identified by initial TBX1 screening, highlighting the limitations of this test as a standalone screening assay.

Abstract 2957: T-cell receptor immunosequencing reveals novel insights into the immune response to human pancreatic cancer

Abstract Introduction Despite advancements in therapy, pancreatic ductal adenocarcinoma (PDA) remains an aggressive cancer with high mortality. It is characterized by dense inflammation, including many T cells; however, it is unclear whether these T cells signify a true antitumor response. In the setting of disappointing early results of immunotherapy in PDA, we sought to gain a deeper understanding of the tumor-associated T-cell response. Methods With IRB approval, we obtained archival resected PDA tumors in paraffin-embedded blocks from 54 patients. We performed histopathology to identify blocks containing lymph nodes (LN) and performed T-cell receptor (TCR) immunosequencing on DNA extracted from immediately adjacent tissue. To address possible intratumoral heterogeneity, we analyzed duplicate samples from separate blocks for 10 patients. Productive clonality was defined as 1-Pielou’s evenness for TCR rearrangements encoding a functional protein; TCR fraction was calculated from number of observed templates and the amount of input DNA. Two-tailed t-tests were used to compare subgroups. TCR sequence overlap between each of the 10 pairs of duplicate samples were calculated at the amino acid level. Results Among samples that did not contain LN, mean TCR fraction was 0.27, and mean clonality was 0.15 (typical peripheral blood mononuclear cell clonality is 0.08). TCR fraction was positively correlated with clonality (R2=0.23, p=0.007), though this correlation was only significant in patients who received neoadjuvant chemotherapy (R2=0.19, p=0.03). TCR fraction was higher in patients with positive nodal status (0.32 vs. 0.23, p=0.02), but lower in patients who received neoadjuvant therapy (0.22 vs. 0.33, p=0.02). There was no survival difference between high or low TCR fraction or clonality. Samples containing LN (not included in previous analyses) had higher TCR fraction (0.49 vs. 0.27, p&amp;lt;0.0001) but lower clonality (0.09 vs. 0.15, p=0.0002); this relationship was maintained regardless of nodal status. Among 10 pairs of duplicate samples, there was 53% mean overlap of TCR amino acid sequences, compared to 0.1% for pairs of unmatched samples (p&amp;lt;0.0001). Conclusion Here we demonstrate evidence of clonal expansion of T cells in the human PDA microenvironment. Extensive overlap of TCR sequences between two distinct samples for each of 10 pairs analyzed further suggests that the clonal expansion may be specific to tumor antigens. Neoadjuvant therapy may also influence clonal expansion, as only treated patients had a positive correlation between TCR fraction and clonality. Finally, the presence of a lymph node appears to skew TCR sequencing data toward a less clonal but more numerous population. This suggests that the most rigorous analysis of TCRs in a solid tumor microenvironment needs to exclude blocks that contain lymph nodes in order to characterize the true intratumoral population of T cells. Citation Format: Yongwoo D. Seo, Florencia G. Jalikis, Xiuyun Jiang, Marissa Vignali, Harlan Robins, Venu G. Pillarisetty. T-cell receptor immunosequencing reveals novel insights into the immune response to human pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2957. doi:10.1158/1538-7445.AM2017-2957