Mature T cell cancers are typically aggressive, treatment resistant and associated with poor prognosis. Clinical application of immunotherapeutic approaches has been limited by a lack of target antigens that discriminate malignant from healthy (normal) T cells. Unlike B cell depletion, pan-T cell aplasia is prohibitively toxic. We report a new targeting strategy based on the mutually exclusive expression of T cell receptor β-chain constant domains 1 and 2 (TRBC1 and TRBC2). We identify an antibody with unique TRBC1 specificity and use it to demonstrate that normal and virus-specific T cell populations contain both TRBC1+ and TRBC2+ compartments, whereas malignancies are restricted to only one. As proof of concept for anti-TRBC immunotherapy, we developed anti-TRBC1 chimeric antigen receptor (CAR) T cells, which recognized and killed normal and malignant TRBC1+, but not TRBC2+, T cells in vitro and in a disseminated mouse model of leukemia. Unlike nonselective approaches targeting the entire T cell population, TRBC-targeted immunotherapy could eradicate a T cell malignancy while preserving sufficient normal T cells to maintain cellular immunity.

OBJECTIVE

More accurate prediction of patient outcome based on molecular subtype is required to identify patients who will benefit from specific treatments.

METHODS

We selected novel 16 candidate prognostic genes, including 10 proliferation-related genes (p-genes) and 6 immune response-related genes (i-genes), from the gene list identified in our previous study. We then analyzed the association between their expression, measured by quantitative real-time reverse transcription-PCR in formalin-fixed, paraffin-embedded tissues, and clinical outcome in 819 breast cancer patients according to molecular subtype.

RESULTS

The prognostic significance of clinical and gene variables varied according to the molecular subtype. Univariate analysis showed that positive lymph node status was significantly correlated with the increased risk of distant metastasis in all subtypes except the hormone receptor-negative, HER2-positive (HR-/HER2+) subtype. Most p-genes were significantly associated with poor prognosis in patients with the HR+/HER2- subtype, whereas i-genes correlated with a favorable outcome in patients with HR-/HER2+ breast cancer. In HR-/HER2+ breast cancer, four genes (three i-genes BTN3A2, CD2, and TRBC1 and the p-gene MMP11) were significantly associated with distant metastasis-free survival (DMFS). A new prognostic model for HR-/HER2+ breast cancer based on the expression of MMP11 and CD2 was developed and the DMFS for patients in the high-risk group according to our model was significantly lower than that for those in the low-risk group. Multivariate analyses revealed that our risk score is an independent prognostic factor for DMFS. Moreover, C-index showed that our risk score has a superior prognostic performance to traditional clinicopathological factors.

CONCLUSIONS

Our new prognostic model for HR-/HER2+ breast cancer provides more accurate information on the risk of distant metastasis than traditional clinical prognostic factors and may be used to identify patients with a good prognosis in this aggressive subtype of breast cancer.

OBJECTIVE

Characterization of altered expression of selected transcripts linked to inflammation in the peripheral blood of sporadic amyotrophic lateral sclerosis (sALS) patients at early stage of disease to increase knowledge about peripheral inflammatory response in sALS.

METHODS

RNA expression levels of 45 genes were assessed by RT-qPCR in 22 sALS cases in parallel with 13 age-matched controls. Clinical and serum parameters were assessed at the same time.

RESULTS

Upregulation of genes coding for factors involved in leukocyte extravasation (ITGB2, INPP5D, SELL, and ICAM1) and extracellular matrix remodeling (MMP9 and TIMP2), as well as downregulation of certain chemokines (CCL5 and CXC5R), anti-inflammatory cytokines (IL10, TGFB2, and IL10RA), pro-inflammatory cytokines (IL-6), and T-cell regulators (CD2 and TRBC1) was found in sALS cases independently of gender, clinical symptoms at onset (spinal, respiratory, or bulbar), progression, peripheral leukocyte number, and integrity of RNA. MMP9 levels positively correlated with age, whereas CCR5, CCL5, and TRBC1 negatively correlated with age in sALS but not in controls. Relatively higher TNFA expression levels correlate with higher creatinine kinase protein levels in plasma.

CONCLUSIONS

Present findings show early inflammatory responses characterized by upregulation of factors enabling extravasation of leukocytes and extracellular matrix remodeling in blood in sALS cases, in addition to increased TNFA levels paralleling skeletal muscle damage.

Objectives: The diagnosis of T-cell large granular lymphocytic leukemia (T-LGLL) is challenging because of overlapping immunophenotypic features with reactive T cells and limitations of T-cell clonality assays. We studied whether adding an antibody against T-cell receptor β constant region 1 (TRBC1) to a comprehensive flow cytometry panel could facilitate the diagnosis of T-LGLL.

Methods: We added TRBC1 antibody to the standard T-cell and natural killer (NK) cell panel to assess T-cell clonality in 56 T-LGLLs and 34 reactive lymphocytoses. In addition, 20 chronic lymphoproliferative disorder of NK cells (CLPD-NKs) and 10 reactive NK-cell lymphocytoses were analyzed.

Results: Clonal T cells were detected in all available T-LGLLs by monotypic TRBC1 expression and clonal/equivocal T-cell receptor gene rearrangement (TCGR) studies, compared with only 27% of T-LGLLs by killer-cell immunoglobulin-like receptor (KIR) restriction. Overall, 85% of T-LGLLs had a blood tumor burden greater than 500 cells/µL. Thirty-four reactive cases showed polytypic TRBC1 expression, except for 5 that revealed small T-cell clones of uncertain significance. All CLPD-NKs showed expected clonal KIR expression and negative TRBC1 expression.

Conclusions: Addition of TRBC1 antibody to the routine flow cytometry assay could replace the TCGR molecular study and KIR flow cytometric analysis to assess clonality, simplifying the diagnosis of T-LGLL.

Keywords: Flow cytometry; T-cell large granular lymphocytic leukemia; T-cell receptor β constant region 1.

T-cell clonality testing is integral to the diagnostic work-up of T-cell malignancies; however, current methods lack specificity and sensitivity, which can make the diagnostic process difficult. The recent discovery of a monoclonal antibody (mAb) specific for human TRBC1 will greatly improve the outlook for T-cell malignancy diagnostics. The anti-TRBC1 mAb can be used in flow cytometry immunophenotyping assays to provide a low-cost, robust, and highly specific test that detects clonality of immunophenotypically distinct T-cell populations. Recent studies demonstrate the clinical utility of this approach in several contexts; use of this antibody in appropriately designed flow cytometry panels improves detection of circulating disease in patients with cutaneous T-cell lymphoma, eliminates the need for molecular clonality testing in the context of large granular lymphocyte leukemia, and provides more conclusive results in the context of many other T-cell disorders. It is worth noting that the increased ability to detect discrete clonal T-cell populations means that identification of T-cell clones of uncertain clinical significance (T-CUS) will become more common. This review discusses this new antibody and describes how it defines clonal T-cells. We present and discuss assay design and summarize findings to date about the use of flow cytometry TRBC1 analysis in the field of diagnostics, including lymph node and fluid sample investigations. We also make suggestions about how to apply the assay results in clinical work-ups, including how to interpret and report findings of T-CUS. Finally, we highlight areas that we think will benefit from further research.

Keywords: T-cell; T-cell receptor; TRBC1; clonality; diagnostics; flow cytometry; leukemia; lymphoma.

Peripheral blood involvement by cutaneous T-cell lymphoma (CTCL) is typically assessed by flow cytometry (FC), and plays a critical role in diagnosis, classification and prognosis. Simplified strategies to detect tumor cells (Sezary cells) fail to exclude reactive subsets, while tumor-specific abnormalities are subtle and inconsistently present. We implemented a FC strategy to detect clonal Sezary cells based on the monotypic expression of one of two mutually exclusive T-cell receptor (TCR) β chain constant regions (TRBCs). Analysis of CD4-positive T-cell subsets and TCR variable β classes from healthy donors showed polytypic TRBC1 staining. Clonal Sezary cells were identified by TRBC1 staining in 56 of 111 (50%) samples from patients with CTCL, accounting for 7 to 18,155 cells/μL, and including 13 cases (23%) lacking tumor-specific immunophenotypic abnormalities. CD4-positive T-cell subsets from 86 patients without T-cell lymphoma showed polytypic TRBC1 staining, except for 5 patients (6%) with minute T-cell clones of uncertain significance (T-CUS) accounting for 53 to 136 cells/μL. Assessment of TRBC1 expression within a comprehensive single-tube FC assay effectively overcomes interpretative uncertainties in the identification of Sezary cells, without the need for a separate T-cell clonality assay.

Keywords: Sezary cell; TRBC1; flow cytometry.

Benign clonal T-cell expansions in reactive immune responses often complicate the laboratory diagnosis T-cell neoplasia. We recently introduced a novel flow cytometry assay to detect T-cell clones in blood and bone marrow, based on the identification of a monophasic T-cell receptor (TCR) β chain constant region-1 (TRBC1) expression pattern within a phenotypically distinct TCRαβ T-cell subset. In routine laboratory practice, T-cell clones of uncertain significance (T-CUS) were detected in 42 of 159 (26%) patients without T-cell malignancy, and in 3 of 24 (13%) healthy donors. Their phenotype (CD8⁺/CD4^-: 78%, CD4^-/CD8^-: 12%, CD4⁺/CD8⁺: 9%, or CD4⁺/CD8^-: 2%) closely resembled that of 26 cases of T-cell large granular lymphocytic leukemia (T-LGLL) studied similarly, except for a much smaller clone size (p < 0.0001), slightly brighter CD2 and CD7, and slightly dimmer CD3 expression (p < 0.05). T-CUS was not associated with age, gender, comorbidities, or peripheral blood counts. TCR-Vβ repertoire analysis confirmed the clonality of T-CUS, and identified additional clonotypic CD8-positive subsets when combined with TRBC1 analysis. We hereby report the phenotypic features and incidence of clonal T-cell subsets in patients with no demonstrable T-cell neoplasia, providing a framework for the differential interpretation of T-cell clones based on their size and phenotypic properties.

T cell therapy represents a new class of immunotherapies garnering considerable attention. T cell receptor beta chain constant region 1 (TRBC1) is partially expressed in subsets of normal T cells. However, the immunotherapy of T lymphocyte tumors is rarely validated in clinical trials. Here, we aim to explore whether TRBC1 is a promising target for the immunotherapy of T lymphocyte tumors. This study examined TRBC1 expression in 25 healthy bone marrow samples, 39 patients with T-lineage acute lymphocytic leukemia (T-ALL), 4 patients with mature T cell neoplasms, and 5 patients suspected with mature T cell neoplasms with evidence of T cell neoplasia. Moreover, the expression of TRBC1 was evaluated by flow cytometry and through PCR detection of TCR gene rearrangements. The expression of monophasic TRBC1 was identified in all 25 normal bone marrows (23.83% ± 2.74% positive rate). The expression of TRBC1 was positive in 5 patients (12.8%) among the 39 T-ALL patients. TRBC1 was partially expressed in 1 patient (25%) with T cell non-Hodgkin's lymphoma (T-NHL) and in 1 patient (20%) suspected to have T-NHL. Healthy donors showed a pattern of partial expression and patients with T-lymphocyte tumors showed a polytypic TRBC1 expression pattern. Thus, TRBC1 may be a diagnostic and therapeutic marker for T lymphocyte tumors.

Keywords: Flow cytometry; T cell receptor rearrangement; T cell receptor β chain constant region 1; T lineage acute lymphocytic leukemia.

Targeting T cell receptor β-chain constant region 1 (TRBC1) CAR-T could specifically kill TRBC1+ T-cell malignancies. However, over-expressed CARs on anti-TRBC1 CAR transduced TRBC1+ T cells (CAR-C1) bound to autologous TRBC1, masking TRBC1 from identification by other anti-TRBC1 CAR-T, and moreover only the remaining unoccupied CARs recognized TRBC1+ cells, considerably reducing therapeutic potency of CAR-C1. In addition, co-culture of anti-TRBC1 CAR-T and TRBC1+ cells could promote exhaustion and terminal differentiation of CAR-T. These findings provide a rationale for pre-depleting TRBC1+ T cells before anti-TRBC1 CAR-T manufacturing.

Keywords: CAR-T; T cell receptor β-chain constant region 1; T-cell malignancy.

Background: The lack of sensitivity and specificity of most biomarkers or the lack of relevant studies to demonstrate their effectiveness in sepsis.

Methods: Downloaded three sets of sepsis expression data (GSE13904, GSE25504, GSE26440) from GEO. Then, using the R limma package and WGCNA analysis tocore genes. Finally, the value of these core genes was confirmed by clinical samples.

Results: Compared to normal samples, we obtain many abnormally expressed genes in the pediatric sepsis. WGCNA co-expression analysis showed that genes from blue and turquoise module were close correlation with pediatric sepsis. The top 20 genes (TIMP2, FLOT1, HCK, NCF4, SERPINA1, IL17RA, PGD, PRKCD, GLT1D1, ALOX5, SIRPA, DOK3, ITGAM, S100A11, ZNF438, PLIN3, LTB4R, TSPO, MAPK14, GAS7) of the blue module of pediatric sepsis were mainly enriched in neutrophil degranulation, etc The top 20 genes (TBC1D4, NOL11, NLRC3, ZNF121, DYRK2, ABCE1, MAGEH1, TMEM263, MCUB, MALT1, DDHD2, TRAC, NOC3L, LCK, TRMT61B, ZNF260, ENOPH1, LOC93622, NAE1, TRBC1) for turquoise module were mainly enriched in rRNA-containing ribonucleoprotein complexes exported from the nucleus, etc The selected hub gene of pediatric sepsis was combined with the markers of cell surface and found 10 core genes (HCK, PRKCD, SIRPA, DOK3, ITGAM, LTB4R, MAPK14, MALT1, NLRC3, LCK). ROC showed that AUC of the 10 core genes for diagnosis of pediatric sepsis was above 0.9.

Conclusion: There were many abnormally expressed genes in patients with pediatric sepsis. The panel constructed by the 10 core genes was expected to become a biomarker panel for clinical application of pediatric sepsis.

Keywords: core gene; diagnosis; panel; pediatric sepsis.

Objectives: Flow cytometric detection of T-cell clonality is challenging, particularly in differential diagnosis of immature T-cell proliferations. Studies have shown utility of TRBC1, in conjunction with other T-cell markers, as reliable means to identify T-cell clonality by flow cytometry. One limitation of surface TRBC1 (sTRBC1) evaluation is it cannot be detected in surface CD3 (sCD3)-negative T cells, such as normal or abnormal immature T-cell precursors. Here, we assess surface and cytoplasmic TRBC1 expression patterns in the differential diagnosis of T-lymphoblastic leukemia/lymphoma (T-ALL) vs normal thymocyte expansions.

Methods: Forty-three samples containing T-ALL, thymoma, normal thymus, and/or indolent T-lymphoblastic proliferation (i-TLBP), were evaluated.

Results: All 24 cases with normal thymocytes or i-TLBPs revealed a characteristic and reproducible sCD3/sTRBC1 expression pattern indicative of polytypic T-cell maturation. In contrast, all 19 T-ALLs lacked this polytypic maturation pattern and were either completely negative for sCD3/sTRBC1 or showed a minor sCD3-positive subset with a monotypic TRBC1 expression pattern. Cytoplasmic TRBC1 evaluation in 9 T-ALLs demonstrated a monotypic intracellular TRBC1-positive (n = 4) or TRBC1-negative (n = 5) expression, indicative of clonality.

Conclusions: Our findings demonstrate flow cytometric evaluation of surface and cytoplasmic TRBC1 expression can aid detection of T-cell clonality and differential diagnosis of immature T-cell proliferations.

Keywords: Flow cytometry; T-ALL; T-cell clonality; TRBC1; Thymoma.

A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor β-chain constant domains was identified as a potentially useful flow-cytometry (FCM) marker to assess Tαβ-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαβ-cells and validated the method in 211 normal, reactive and pathological samples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1⁺ and TRBC1^- monoclonal and polyclonal Tαβ-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1⁺/TRBC1^- ratios within different Tαβ-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVβ families, whereas highly-variable TRBC1⁺/TRBC1^- ratios were observed in more mature vs. naïve Tαβ-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal Tαβ-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαβ-cells of ≤10^-4 in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature Tαβ⁺ leukemia/lymphoma patients.

Keywords: JOVI-1; MRD1; T-CLPD; TCRVβ; TRBC1; TRBJ1 and TRBJ2; Tαβ-cells.

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of autoimmune origin, in which inflammation and demyelination lead to neurodegeneration and progressive disability. Treatment is aimed at slowing down the course of the disease and mitigating its symptoms. One of the first-line treatments used in patients with MS is glatiramer acetate (GA). However, in clinical practice, a response rate of between 30% and 55% is observed. This variability in the effectiveness of the medication may be influenced by genetic factors such as polymorphisms in the genes involved in the pathogenesis of MS. Therefore, this review assesses the impact of genetic variants on the response to GA therapy in patients diagnosed with MS. The results suggest that a relationship exists between the effectiveness of the treatment with GA and the presence of polymorphisms in the following genes: CD86, CLEC16A, CTSS, EOMES, MBP, FAS, TRBC1, IL1R1, IL12RB2, IL22RA2, PTPRT, PVT1, ALOX5AP, MAGI2, ZAK, RFPL3, UVRAG, SLC1A4, and HLA-DRB1*1501. Consequently, the identification of polymorphisms in these genes can be used in the future as a predictive marker of the response to GA treatment in patients diagnosed with MS. Nevertheless, there is a lack of evidence for this and more validation studies need to be conducted to apply this information to clinical practice.

Keywords: glatiramer acetate; medicine personalized; multiple sclerosis; myelin basic protein; pharmacogenetics; polymorphisms; response; treatment.

Peripheral T-cell lymphoma (PTCL) is a type of non-Hodgkin lymphoma that progresses aggressively with poor survival rate. CAR T cell targeting T-cell receptor β-chain constant domains 1 (TRBC1) of malignant T cells has been developed recently by using JOVI.1 monoclonal antibody as a template. However, the mode of JOVI.1 binding is still unknown. This study aimed to investigate the molecular interaction between JOVI.1 antibody and TRBC1 by using computational methods and molecular docking. Therefore, the TRBC protein crystal structures (TRBC1 and TRBC2) as well as the sequences of JOVI.1 CDR were chosen as the starting materials. TRBC1 and TRBC2 epitopes were predicted, and molecular dynamic (MD) simulation was used to visualize the protein dynamic behavior. The structure of JOVI.1 antibody was also generated before the binding mode was predicted using molecular docking with an antibody mode. Epitope prediction suggested that the N3K4 region of TRBC1 may be a key to distinguish TRBC1 from TCBC2. MD simulation showed the major different surface conformation in this area between two TRBCs. The JOVI.1-TRBC1 structures with three binding modes demonstrated JOVI.1 interacted TRBC1 at N3K4 residues, with the predicted dissociation constant (K_d) ranging from 1.5 × 10⁸ to 1.1 × 10¹⁰ M. The analysis demonstrated JOVI.1 needed D1 residues of TRBC1 for the interaction formation to N3K4 in all binding modes. In conclusion, we proposed the three binding modes of the JOVI.1 antibody to TRBC1 with the new key residue (D1) necessary for N3K4 interaction. This data was useful for JOVI.1 redesign to improve the PTCL-targeting CAR T cell.

Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor β chain (TRBC1) expression for assessing Tαβ-cell clonality has been recently validated. However, its utility for the diagnosis of clonality of T-large granular lymphocytic leukemia (T-LGLL) needs to be confirmed, since more mature Tαβ cells (i.e., T-LGL normal-counterpart) show broader TRBC1⁺/TRBC1^- ratios vs. total Tαβ cells. We compared the distribution and absolute counts of TRBC1⁺ and TRBC1^- Tαβ-LGL in blood containing polyclonal (n = 25) vs. clonal (n = 29) LGL. Overall, polyclonal TRBC1⁺ or TRBC1^- Tαβ-LGL ranged between 0.36 and 571 cells/μL (3.2-91% TRBC1⁺ cells), whereas the clonal LGL cases showed between 51 and 11,678 cells/μL (<0.9% or >96% TRBC1⁺ cells). Among the distinct TCRVβ families, the CD28^- effector-memory and terminal-effector polyclonal Tαβ cells ranged between 0 and 25 TRBC1⁺ or TRBC1^- cells/μL and between 0 and 100% TRBC1⁺ cells, while clonal LGL ranged between 32 and 5515 TRBC1⁺ or TRBC1^- cells/μL, representing <1.6% or >98% TRBC1⁺ cells. Our data support the utility of the TRBC1-FCM assay for detecting T-cell clonality in expansions of Tαβ-LGL suspected of T-LGLL based on altered percentages of TRBC1⁺ Tαβ cells. However, in the absence of lymphocytosis or in the case of TαβCD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of Tαβ-LGL-expressing individual TCRVβ families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations.

Keywords: JOVI-1; LGL; LGLL; TCRVβ repertoire; TRBC1; Tαβ effector cells; Tαβ-cell maturation stages; flow cytometry T-cell clonality assessment; large granular lymphocytes; large granular lymphocytic leukemia.