Peripheral blood T-cell receptor repertoire as a predictor of clinical outcomes in gastrointestinal cancer patients treated with PD-1 inhibitor

Background Identifying valid biomarkers for patient selection impressively promotes the success of anti-PD-1 therapy. However, the unmet need for biomarkers in gastrointestinal (GI) cancers remains significant. We aimed to explore the predictive value of the circulating T-cell receptor (TCR) repertoire for clinical outcomes in GI cancers who received anti-PD-1 therapy. Methods 137 pre- and 79 post-treated peripheral blood samples were included. The TCR repertoire was evaluated by sequencing of complementarity-determining region 3 (CDR3) in the TRB gene. The Shannon index was used to measure the diversity of the TCR repertoire, and Morisita’s overlap index was used to determine TCR repertoire similarities between pre- and post-treated samples. Results Among all enrolled patients, 76 received anti-PD-1 monotherapy and 61 received anti-PD-1 combination therapy. In the anti-PD-1 monotherapy cohort, patients with higher baseline TCR diversity exhibited a significantly higher disease control rate (77.8% vs. 47.2%; hazard ratio [HR] 3.92; 95% confidence interval [CI] 1.14–13.48; P = 0.030) and a longer progression-free survival (PFS) (median: 6.47 months vs. 2.77 months; HR 2.10; 95% CI 1.16–3.79; P = 0.014) and overall survival (OS) (median: NA vs. 8.97 months; HR 3.53; 95% CI 1.49–8.38; P = 0.004) than those with lower diversity. Moreover, patients with a higher TCR repertoire similarity still showed a superior PFS (4.43 months vs. 1.84 months; HR 13.98; 95% CI 4.37–44.68; P < 0.001) and OS (13.40 months vs. 6.12 months; HR 2.93; 95% CI 1.22–7.03; P = 0.016) even in the cohort with lower baseline diversity. However, neither biomarker showed predictive value in the anti-PD-1 combination therapy cohort. Interestingly, the combination of TCR diversity and PD-L1 expression can facilitate patient stratification in a pooled cohort. Conclusion The circulating TCR repertoire can serve as a predictor of clinical outcomes in anti-PD-1 therapy in GI cancers (AU)

TCR repertoire diversity in Multiple Sclerosis: High-dimensional bioinformatics analysis of sequences from brain, cerebrospinal fluid and peripheral blood.

BACKGROUND: T cells play a key role in the pathogenesis of multiple sclerosis (MS), a chronic, inflammatory, demyelinating disease of the central nervous system (CNS). Although several studies recently investigated the T-cell receptor (TCR) repertoire in cerebrospinal fluid (CSF) of MS patients by high-throughput sequencing (HTS), a deep analysis on repertoire similarities and differences among compartments is still missing. METHODS: We performed comprehensive bioinformatics on high-dimensional TCR Vß sequencing data from published and unpublished MS and healthy donors (HD) studies. We evaluated repertoire polarization, clone distribution, shared CDR3 amino acid sequences (CDR3s-a.a.) across repertoires, clone overlap with public databases, and TCR similarity architecture. FINDINGS: CSF repertoires showed a significantly higher public clones percentage and sequence similarity compared to peripheral blood (PB). On the other hand, we failed to reject the null hypothesis that the repertoire polarization is the same between CSF and PB. One Primary-Progressive MS (PPMS) CSF repertoire differed from the others in terms of TCR similarity architecture. Cluster analysis splits MS from HD. INTERPRETATION: In MS patients, the presence of a physiological barrier, the blood-brain barrier, does not impact clone prevalence and distribution, but impacts public clones, indicating CSF as a more private site. We reported a high Vß sequence similarity in the CSF-TCR architecture in one PPMS. If confirmed it may be an interesting insight into MS progressive inflammatory mechanisms. The clustering of MS repertoires from HD suggests that disease shapes the TCR Vß clonal profile. FUNDING: This study was partly financially supported by the Italian Multiple Sclerosis Foundation (FISM), that contributed to Ballerini-DB data collection (grant #2015 R02).

Persistent T Cell Repertoire Perturbation and T Cell Activation in HIV After Long Term Treatment.

Objective: In people living with HIV (PLHIV), we sought to test the hypothesis that long term anti-retroviral therapy restores the normal T cell repertoire, and investigate the functional relationship of residual repertoire abnormalities to persistent immune system dysregulation. Methods: We conducted a case-control study in PLHIV and HIV-negative volunteers, of circulating T cell receptor repertoires and whole blood transcriptomes by RNA sequencing, complemented by metadata from routinely collected health care records. Results: T cell receptor sequencing revealed persistent abnormalities in the clonal T cell repertoire of PLHIV, characterized by reduced repertoire diversity and oligoclonal T cell expansion correlated with elevated CD8 T cell counts. We found no evidence that these expansions were driven by cytomegalovirus or another common antigen. Increased frequency of long CDR3 sequences and reduced frequency of public sequences among the expanded clones implicated abnormal thymic selection as a contributing factor. These abnormalities in the repertoire correlated with systems level evidence of persistent T cell activation in genome-wide blood transcriptomes. Conclusions: The diversity of T cell receptor repertoires in PLHIV on long term anti-retroviral therapy remains significantly depleted, and skewed by idiosyncratic clones, partly attributable to altered thymic output and associated with T cell mediated chronic immune activation. Further investigation of thymic function and the antigenic drivers of T cell clonal selection in PLHIV are critical to efforts to fully re-establish normal immune function.

Peripheral blood T-cell receptor repertoire as a predictor of clinical outcomes in gastrointestinal cancer patients treated with PD-1 inhibitor.

BACKGROUND: Identifying valid biomarkers for patient selection impressively promotes the success of anti-PD-1 therapy. However, the unmet need for biomarkers in gastrointestinal (GI) cancers remains significant. We aimed to explore the predictive value of the circulating T-cell receptor (TCR) repertoire for clinical outcomes in GI cancers who received anti-PD-1 therapy. METHODS: 137 pre- and 79 post-treated peripheral blood samples were included. The TCR repertoire was evaluated by sequencing of complementarity-determining region 3 (CDR3) in the TRB gene. The Shannon index was used to measure the diversity of the TCR repertoire, and Morisita's overlap index was used to determine TCR repertoire similarities between pre- and post-treated samples. RESULTS: Among all enrolled patients, 76 received anti-PD-1 monotherapy and 61 received anti-PD-1 combination therapy. In the anti-PD-1 monotherapy cohort, patients with higher baseline TCR diversity exhibited a significantly higher disease control rate (77.8% vs. 47.2%; hazard ratio [HR] 3.92; 95% confidence interval [CI] 1.14-13.48; P = 0.030) and a longer progression-free survival (PFS) (median: 6.47 months vs. 2.77 months; HR 2.10; 95% CI 1.16-3.79; P = 0.014) and overall survival (OS) (median: NA vs. 8.97 months; HR 3.53; 95% CI 1.49-8.38; P = 0.004) than those with lower diversity. Moreover, patients with a higher TCR repertoire similarity still showed a superior PFS (4.43 months vs. 1.84 months; HR 13.98; 95% CI 4.37-44.68; P < 0.001) and OS (13.40 months vs. 6.12 months; HR 2.93; 95% CI 1.22-7.03; P = 0.016) even in the cohort with lower baseline diversity. However, neither biomarker showed predictive value in the anti-PD-1 combination therapy cohort. Interestingly, the combination of TCR diversity and PD-L1 expression can facilitate patient stratification in a pooled cohort. CONCLUSION: The circulating TCR repertoire can serve as a predictor of clinical outcomes in anti-PD-1 therapy in GI cancers.

Leukemic extracellular vesicles induce chimeric antigen receptor T cell dysfunction in chronic lymphocytic leukemia.

Chimeric antigen receptor (CAR) T cell therapy has yielded unprecedented outcomes in some patients with hematological malignancies; however, inhibition by the tumor microenvironment has prevented the broader success of CART cell therapy. We used chronic lymphocytic leukemia (CLL) as a model to investigate the interactions between the tumor microenvironment and CART cells. CLL is characterized by an immunosuppressive microenvironment, an abundance of systemic extracellular vesicles (EVs), and a relatively lower durable response rate to CART cell therapy. In this study, we characterized plasma EVs from untreated CLL patients and identified their leukemic cell origin. CLL-derived EVs were able to induce a state of CART cell dysfunction characterized by phenotypical, functional, and transcriptional changes of exhaustion. We demonstrate that, specifically, PD-L1+ CLL-derived EVs induce CART cell exhaustion. In conclusion, we identify an important mechanism of CART cell exhaustion induced by EVs from CLL patients.

[First universal newborn screening program for severe combined immunodeficiency in Europe. Three-years' experience in Catalonia.] / Primer programa europeo de cribado neonatal para la inmunodeficiencia combinada grave: experiencia de tres años en Cataluña.

Severe combined immunodeficiency (SCID), the most severe form of T-cell immunodeficiency, can be screened at birth by quantifying T-cell receptor excision circles (TREC) in dried blood spot (DBS) samples. Early detection of this condition speeds up the establishment of appropriate treatment and increases the patient's life expectancy. Newborn screening for SCID started in January 2017 in Catalonia, the first Spanish and European region to universally include this testing. The results obtained in the first three years and a half of experience (January 2017 - June 2020) are shown here, using EnLite Neonatal TREC kit (Perkin Elmer) with 20 copies/µL as TREC detection cutoff. Of 222,857 newborns screened, 48 tested positive: three patients were diagnosed with SCID (incidence 1:74,285); 17 patients had clinically significant T-cell lymphopenia (non-SCID) with an incidence of 1 in 13,109 newborns; twenty two patients were considered false-positive cases because of an initially normal lymphocyte count with normalization of TREC between 3 and 6 months of life; one case had transient lymphopenia due to an initially low lymphocyte count with recovery in the following months; and five patients are still under study. The results obtained provide further evidence of the benefits of including this disease in newborn screening programs. Even longer follow-up could be necessary to define the exact incidence of SCID in Catalonia.

La inmunodeficiencia combinada grave (IDCG) es la forma más grave de inmunodeficiencia primaria, que afecta sobre todo a los linfocitos T, y puede ser detectada al nacer mediante la cuantificación de los círculos de escisión del receptor de linfocitos T (TREC) en una muestra de sangre impregnada en papel (DBS). La detección precoz de esta enfermedad permite establecer de forma temprana un tratamiento adecuado en el paciente, permitiendo así su curación. El cribado neonatal de IDCG comenzó en Cataluña en enero de 2017, siendo la primera región española y europea en incluirla oficial y universalmente en su programa. En el presente trabajo se presentan los resultados obtenidos durante los tres primeros años y medio de experiencia (enero 2017 ­ junio 2020) empleando el kit EnLite Neonatal TREC (Perkin Elmer), con un cutoff de detección de TREC de 20 copias/µL. De 222.857 recién nacidos analizados, cuarenta y ocho fueron detecciones positivas: tres casos de IDCG (incidencia de 1:74.285); diecisiete casos de linfopenia T no IDCG (incidencia de 1:13.109); veintidós casos falsos positivos (recuento de linfocitos inicialmente normal, con normalización de TREC entre los tres y seis meses de vida); un caso con linfopenia transitoria (con un recuento de linfocitos inicialmente bajo, que se normaliza en los meses siguientes); y cinco pacientes se encuentran todavía en estudio. Los resultados obtenidos aportan evidencias de los beneficios que supone incluir esta enfermedad en los programas de cribado neonatal. Podría ser necesario un seguimiento todavía más prolongado para acabar de definir la incidencia exacta de IDCG en Cataluña.

Primer programa europeo de cribado neonatal para la inmunodeficiencia combinada grave: experiencia de tres años en Cataluña / First universal newborn screening program for severe combined immunodeficiency in Europe. Three-years' experience in Catalonia

La inmunodeficiencia combinada grave (IDCG) es la forma más grave de inmunodeficiencia primaria, que afecta sobre todo a los linfocitos T, y puede ser detectada al nacer mediante la cuantificación de los círculos de escisión del receptor de linfocitos T (TREC) en una muestra de sangre impregnada en papel (DBS). La detección precoz de esta enfermedad permite establecer de forma temprana un tratamiento adecuado en el paciente, permitiendo así su curación. El cribado neonatal de IDCG comenzó en Cataluña en enero de 2017, siendo la primera región española y europea en incluirla oficial y universalmente en su programa. En el presente trabajo se presentan los resultados obtenidos durante los tres primeros años y medio de experiencia (enero 2017 - junio 2020) empleando el kit EnLite Neonatal TREC (Perkin Elmer), con un cutoff de detección de TREC de 20 copias/miL. De 222.857 recién nacidos analizados, cuarenta y ocho fueron detecciones positivas: tres casos de IDCG (incidencia de 1:74.285); diecisiete casos de linfopenia T no IDCG (incidencia de 1:13.109); veintidós casos falsos positivos (recuento de linfocitos inicialmente normal, con normalización de TREC entre los tres y seis meses de vida); un caso con linfopenia transitoria (con un recuento de linfocitos inicialmente bajo, que se normaliza en los meses siguientes); y cinco pacientes se encuentran todavía en estudio. Los resultados obtenidos aportan evidencias de los beneficios que supone incluir esta enfermedad en los programas de cribado neonatal. Podría ser necesario un seguimiento todavía más prolongado para acabar de definir la incidencia exacta de IDCG en Cataluña

Severe combined immunodeficiency (SCID), the most severe form of T-cell immunodeficiency, can be screened at birth by quantifying T-cell receptor excision circles (TREC) in dried blood spot (DBS) samples. Early detection of this condition speeds up the establishment of appropriate treatment and increases the patient's life expectancy. Newborn screening for SCID started in January 2017 in Catalonia, the first Spanish and European region to universally include this testing. The results obtained in the first three years and a half of experience (January 2017 - June 2020) are shown here, using EnLite Neonatal TREC kit (Perkin Elmer) with 20 copies/miL as TREC detection cutoff. Of 222,857 newborns screened, 48 tested positive: three patients were diagnosed with SCID (incidence 1:74,285); 17 patients had clinically significant T-cell lymphopenia (non-SCID) with an incidence of 1 in 13,109 newborns; twenty two patients were considered false-positive cases because of an initially normal lymphocyte count with normalization of TREC between 3 and 6 months of life; one case had transient lymphopenia due to an initially low lymphocyte count with recovery in the following months; and five patients are still under study. The results obtained provide further evidence of the benefits of including this disease in newborn screening programs. Even longer follow-up could be necessary to define the exact incidence of SCID in Catalonia

Next generation sequencing based assessment of the alloreactive T cell receptor repertoire in kidney transplant patients during rejection: a prospective cohort study.

BACKGROUND: Kidney transplantation is the optimal treatment in end stage renal disease but the allograft survival is still hampered by immune reactions against the allograft. This process is driven by the recognition of allogenic antigens presented to T-cells and their unique T-cell receptor (TCR) via the major histocompatibility complex (MHC), which triggers a complex immune response potentially leading to graft injury. Although the immune system and kidney transplantation have been studied extensively, the subtlety of alloreactive immune responses has impeded sensitive detection at an early stage. Next generation sequencing of the TCR enables us to monitor alloreactive T-cell populations and might thus allow the detection of early rejection events. METHODS/DESIGN: This is a prospective cohort study designed to sequentially evaluate the alloreactive T cell repertoire after kidney transplantation. The TCR repertoire of patients who developed biopsy confirmed acute T cell mediated rejection (TCMR) will be compared to patients without rejection. To track the alloreactive subsets we will perform a mixed lymphocyte reaction between kidney donor and recipient before transplantation and define the alloreactive TCR repertoire by next generation sequencing of the complementary determining region 3 (CDR3) of the T cell receptor beta chain. After initial clonotype assembly from sequencing reads, TCR repertoire diversity and clonal expansion of T cells of kidney transplant recipients in periphery and kidney biopsy will be analyzed for changes after transplantation, during, prior or after a rejection. The goal of this study is to describe changes of overall T cell repertoire diversity, clonality in kidney transplant recipients, define and track alloreactive T cells in the posttransplant course and decipher patterns of expanded alloreactive T cells in acute cellular rejection to find an alternative monitoring to invasive and delayed diagnostic procedures. DISCUSSION: Changes of the T cell repertoire and tracking of alloreactive T cell clones after combined bone marrow and kidney transplant has proven to be of potential use to monitor the donor directed alloresponse. The dynamics of the donor specific T cells in regular kidney transplant recipients in rejection still rests elusive and can give further insights in human alloresponse. TRIAL REGISTRATION: Clinicaltrials.gov: NCT03422224 , registered February 5th 2018.

Comparison between flow cytometry and standard PCR in the evaluation of MRD in children with acute lymphoblastic leukemia treated with the GBTLI LLA - 2009 protocol.

Minimal residual disease (MRD) monitoring is of prognostic importance in childhood acute lymphoblastic leukemia (ALL). The detection of immunoglobulin and T-cell receptor gene rearrangements by real-time quantitative PCR (RT-PCR) is considered the gold standard for this evaluation. However, more accessible methods also show satisfactory performance. This study aimed to compare MRD analysis by four-color flow cytometry (FC) and qualitative standard PCR on days 35 and 78 of chemotherapy and to correlate these data with patients' clinical characteristics. Forty-two children with a recent diagnosis of ALL, admitted to a public hospital in Brazil for treatment in accordance with the Brazilian Childhood Cooperative Group for ALL Treatment (GBTLI LLA-2009), were included. Bone marrow samples collected at diagnosis and on days 35 and 78 of treatment were analyzed for the immunophenotypic characterization of blasts by FC and for the detection of clonal rearrangements by standard PCR. Paired analyses were performed in 61/68 (89.7%) follow-up samples, with a general agreement of 88.5%. Disagreements were resolved by RT-PCR, which evidenced one false-negative and four false-positive results in FC, as well as two false-negative results in PCR. Among the prognostic factors, a significant association was found only between T-cell lineage and MRD by standard PCR. These results show that FC and standard PCR produce similar results in MRD detection of childhood ALL and that both methodologies may be useful in the monitoring of disease treatment, especially in regions with limited financial resources.

T-Cell Receptor Excision Circles in Newborns with Congenital Heart Disease.

OBJECTIVES: To determine if children with congenital heart disease (CHD) have lower newborn T-cell receptor excision circles (TREC) levels than the general population and to evaluate if low TREC levels in newborns with CHD are associated with clinical complications such as hospitalization for infection. STUDY DESIGN: The Connecticut Newborn Screening Program reported TREC levels for newborns with CHD delivered between October 2011 and September 2016 at 2 major Connecticut children's hospitals. TREC levels for children with CHD were compared with the general population. TREC levels and outcome measures, including hospitalization for infection, were compared. RESULTS: We enrolled 575 participants with CHD in the study. The median TREC level for newborns with CHD was lower than the general population (180.1 copies/µL vs 312.5 copies/µL; P < .01). patients with CHD requiring hospitalization for infection had lower median TREC levels than their counterparts (143.0 copies/µL vs 186.7 copies/µL; P < .01). The combination of prematurity and low TREC level had a strong relationship to hospitalization for infection (area under the receiver operative characteristic curve of 0.89). There was no association between TREC level and CHD severity. CONCLUSIONS: Newborns with CHD demonstrated lower TREC levels than the general population. Low TREC levels were associated with hospitalization for infection in preterm children with CHD. Study limitations include that this was a retrospective chart review. These findings may help to identify newborns with CHD at highest risk for infection, allowing for potential opportunities for intervention.

Reference intervals for lymphocyte subsets in preterm and term neonates without immune defects.

BACKGROUND: In 6.5 years of newborn screening for severe combined immunodeficiency in California, 3,252,156 infants had DNA from dried blood spots (DBSs) assayed for T-cell receptor excision circles. Infants with T-cell receptor excision circle values of less than a designated cutoff on a single DBS, 2 DBS samples with insufficient PCR amplification, or known genetic risk of immunodeficiency had peripheral blood complete blood counts and lymphocyte subsets assayed in a single flow cytometry laboratory. Cases in which immune defects were ruled out were available for analysis. OBJECTIVE: We sought to determine reference intervals for lymphocyte subsets in racially/ethnically diverse preterm and term newborns who proved to be unaffected by any T-lymphopenic immune disorder. METHODS: Effective gestational age (GA) was defined as GA at birth plus postnatal age at the time of sample collection. After determining exclusion criteria, we analyzed demographic and clinical information, complete and differential white blood cell counts, and lymphocyte subsets for 301 infants, with serial measurements for 33 infants. Lymphocyte subset measurements included total T cells, helper and cytotoxic T-cell subsets, naive and memory phenotype of each T-cell subset, B cells, and natural killer cells. RESULTS: Reference intervals were generated for absolute numbers and lymphocyte subsets from infants with effective GAs of 22 to 52 weeks. Sex and ethnicity were not significant determinants of lymphocyte subset counts in this population. Lymphocyte counts increased postnatally. CONCLUSION: This study provides a baseline for interpreting comprehensive lymphocyte data in preterm and term infants, aiding clinicians to determine which newborns require further evaluations for immunodeficiency.

Factors predicting the recurrence of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in children after treatment using the HLH-2004 protocol.

Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) is highly prevalent in Japan. To date, no standard treatment for EBV-HLH has been established owing to the diversity in treatment response and the difficulty in assessing prognostic factors. The present prospective study recruited 27 children with EBV-HLH who were also part of the HLH-2004 study. EBV load in the peripheral blood was monitored at diagnosis and 2, 4, and 8 weeks after treatment initiation. Additionally, T-cell receptor (TCR) clonality and other laboratory data were evaluated. TCR clonality was positive in 14 patients at diagnosis. Seven of 27 patients experienced recurrences after treatment. No correlation was noted among any clinical data at diagnosis of patients with and without recurrence. However, the recurrence rate was significantly higher in patients aged < 2 years and/or those with a high plasma EBV load of > 103 copies/mL 2 weeks after treatment than that in patients without these factors. These findings suggest that a younger age or a high EBV load in plasma at the early phase of treatment is a factor predicting a recurrence and helps guide the intensity of subsequent treatment phases for children with EBV-HLH.

Characteristics and prognostic significance of profiling the peripheral blood T-cell receptor repertoire in patients with advanced lung cancer.

Lung cancer is one of the greatest threats to human health, and is initially detected and attacked by the immune system through tumor-reactive T cells. The aim of this study was to determine the basic characteristics and clinical significance of the peripheral blood T-cell receptor (TCR) repertoire in patients with advanced lung cancer. To comprehensively profile the TCR repertoire, high-throughput sequencing was used to identify hypervariable rearrangements of complementarity determining region 3 (CDR3) of the TCR ß chain in peripheral blood samples from 64 advanced lung cancer patients and 31 healthy controls. We found that the TCR repertoire differed substantially between lung cancer patients and healthy controls in terms of CDR3 clonotype, diversity, V/J segment usage, and sequence. Specifically, baseline diversity correlated with several clinical characteristics, and high diversity reflected a better immune status. Dynamic detection of the TCR repertoire during anticancer treatment was useful for prognosis. Both increased diversity and high overlap rate between the pre- and post-treatment TCR repertoires indicated clinical benefit. Combination of the diversity and overlap rate was used to categorize patients into immune improved or immune worsened groups and demonstrated enhanced prognostic significance. In conclusion, TCR repertoire analysis served as a useful indicator of disease development and prognosis in advanced lung cancer and may be utilized to direct future immunotherapy.

TCR Repertoire as a Novel Indicator for Immune Monitoring and Prognosis Assessment of Patients With Cervical Cancer.

There is increasing evidence that deep sequencing-based T cell repertoire can sever as a biomarker of immune response in cancer patients; however, the characteristics of T cell repertoire including diversity and similarity, as well as its prognostic significance in patients with cervical cancer (CC) remain unknown. In this study, we applied a high throughput T cell receptor (TCR) sequencing method to characterize the T cell repertoires of peripheral blood samples from 25 CC patients, 30 cervical intraepithelial neoplasia (CIN) patients and 20 healthy women for understanding the immune alterations during the cervix carcinogenesis. In addition, we also explored the signatures of TCR repertoires in the cervical tumor tissues and paired sentinel lymph nodes from 16 CC patients and their potential value in predicting the prognosis of patients. Our results revealed that the diversity of circulating TCR repertoire gradually decreased during the cervix carcinogenesis and progression, but the circulating TCR repertoires in CC patients were more similar to CIN patients than healthy women. Interestingly, several clonotypes uniquely detected in CC patients tended to share similar CDR3 motifs, which differed from those observed in CIN patients. In addition, the TCR repertoire diversity in sentinel lymphatic nodes from CC patients was higher than in tumor tissues. More importantly, less clonotypes in TCR repertoire of sentinel lymphatic node was associated with the poor prognosis of the patients. Overall, our findings suggested that TCR repertoire might be a potential indicator of immune monitoring and a biomarker for predicting the prognosis of CC patients. Although functional studies of T cell populations are clearly required, this study have expanded our understanding of T cell immunity during the development of CC and provided an experimental basis for further studies on its pathogenesis and immunotherapy.

Dysregulated circRNAs in plasma from active tuberculosis patients.

Endogenous circular RNAs (circRNAs) have been reported in various diseases. However, their role in active TB remains unknown. The study was aimed to determine plasma circRNA expression profile to characterize potential biomarker and improve our understanding of active TB pathogenesis. CircRNA expression profiles were screened by circRNA microarrays in active TB plasma samples. Dysregulated circRNAs were then verified by qRT-PCR. CircRNA targets were predicted based on analysis of circRNA-miRNA-mRNA interaction. GO and KEGG pathway analyses were used to predict the function of circRNA. ROC curve was calculated to evaluate diagnostic value for active TB. A total of 75 circRNAs were significantly dysregulated in active TB plasma. By further validation, hsa_circRNA_103571 exhibited significant decrease in active TB patients and showed potential interaction with active TB-related miRNAs such as miR-29a and miR-16. Bioinformatics analysis revealed that hsa_circRNA_103571 was primarily involved in ras signalling pathway, regulation of actin cytoskeleton, T- and B-cell receptor signalling pathway. ROC curve analysis suggested that hsa_circRNA_103571 had significant value for active TB diagnosis. Circulating circRNA dysregulation may play a role in active TB pathogenesis. Hsa_circRNA_103571 may be served as a potential biomarker for active TB diagnosis, and hsa_circRNA_103571-miRNA-mRNA interaction may provide some novel mechanism for active TB.

Lymphocyte apheresis for chimeric antigen receptor T-cell manufacturing in children and young adults with leukemia and neuroblastoma.

BACKGROUND: The first step in the production of chimeric antigen receptor T cells is the collection of autologous T cells using apheresis technology. The procedure is technically challenging, because patients often have low leukocyte counts and are heavily pretreated with multiple lines of chemotherapy, marrow transplantation, and/or radiotherapy. Here, we report our experience of collecting T lymphocytes for chimeric antigen receptor T-cell manufacturing in pediatric and young adult patients with leukemia, non-Hodgkin lymphoma, or neuroblastoma. STUDY DESIGN AND METHODS: Apheresis procedures were performed on a COBE Spectra machine using the mononuclear cell program, with a collection target of 1 × 109 total mononuclear cells per kilogram. Data were collected regarding preapheresis and postapheresis blood counts, apheresis parameters, products, and adverse events. RESULTS: Ninety-nine patients (ages 1.3-25.7 years) and 102 apheresis events were available for analysis. Patients underwent apheresis at a variety of absolute lymphocyte cell counts, with a median absolute lymphocyte count of 944 cells/µL (range, 142-6944 cells/µL). Twenty-two patients (21.6%) had absolute lymphocyte counts less than 500 cells/µL. The mononuclear cell target was obtained in 100% of all apheresis harvests, and chimeric antigen receptor T-cell production was possible from the majority of collections (94%). Mononuclear cell collection efficiency was 65.4%, and T-lymphocyte collection efficiency was 83.4%. Ten patients (9.8%) presented with minor adverse events during the 102 apheresis procedures, with one exception of a severe allergy. CONCLUSIONS: Mononuclear cell apheresis for chimeric antigen receptor T-cell therapy is well tolerated and safe, and it is possible to obtain an adequate quantity of CD3+ lymphocytes for chimeric antigen receptor T-cell manufacturing in heavily pretreated patients who have low lymphocyte counts.

Establishment of a Quantitative Polymerase Chain Reaction Assay for Monitoring Chimeric Antigen Receptor T Cells in Peripheral Blood.

BACKGROUND: The chimeric antigen receptor (CAR) consists of an antigen recognition moiety from a monoclonal antibody fused to an intracellular signalling domain capable of activating T cells. The specific structure of the CAR molecule has been used in various basic research and clinical settings to detect CAR expression, but it is necessary to develop more specific and simpler monitoring methods to observe real-time changes. MATERIALS AND METHODS: To develop a quantitative assay for the universal detection of DNA from anti-CD19 CAR-T cells, a TaqMan real-time quantitative polymerase chain reaction (qPCR) assay was developed using primers based on FMC63-28Z gene sequences. We identified the numbers of copies of CAR gene on T cells transduced with the CAR gene that were obtained from peripheral blood. RESULTS: The assay had a minimum detection limit of 10 copies/µL and a strong linear standard curve (y = -3.3682x + 38.594; R2 = 0.999) within the range of the input CAR gene (10-107 copies/µL). The reproducibility test showed a coefficient of variation ranging from 0.63%-1.65%. Real-time qPCR is a highly sensitive, specific, reproducible, and universal method that can be used to detect anti-CD19 CAR-T cells in peripheral blood.

Newborn screening for severe combined immunodeficiency: Evaluation of a commercial T-cell receptor excision circle-based method in Victorian dried blood spots.

AIM: Severe combined immunodeficiency (SCID) is the most severe form of primary immunodeficiency and is fatal in infancy if untreated. As early diagnosis is associated with improved outcomes, SCID is an ideal condition to consider for inclusion in a newborn screening (NBS) programme in Australia. In this feasibility study, we evaluated the EnLite Neonatal TREC kit for detection of T-cell receptor excision circles (TRECs) from NBS dried blood spots for the identification of known SCID patients in Victoria. METHODS: TREC copies/µL were measured retrospectively in 14 children diagnosed with SCID or complete DiGeorge syndrome (CDGS) from 2005 to 2015 at the Royal Children's Hospital, Melbourne. In addition, TREC copies/µL were measured for 501 prospective de-identified NBS cards. RESULTS: Of 14 known SCID or CDGS samples, 11 were correctly identified as presumptive positive samples with low or undetectable TREC on duplicate testing. The remaining three samples also had low or undetectable TREC on duplicate testing but were considered invalid due to insufficient ß-actin DNA amplification. Of the 501 prospective NBS samples, none were identified as presumptive positive samples on duplicate testing. CONCLUSIONS: The EnLite Neonatal TREC kit correctly identified known SCID or CDGS patients as presumptive positive samples, and initial cut-offs for TREC and ß-actin in the Victorian NBS population were determined. A larger pilot study is required to confirm these proposed cut-offs and to evaluate the cost and implementation of this screening programme in Victoria, Australia. Overall, this study provides preliminary data to support the introduction of this assay to the NBS programme in Victoria.