RAG gene defects at the verge of immunodeficiency and immune dysregulation

Mutations of the recombinase activating genes (RAG) in humans underlie a broad spectrum of clinical and immunological phenotypes that reflect different degrees of impairment of T‐ and B‐cell development and alterations of mechanisms of central and peripheral tolerance. Recent studies have shown that this phenotypic heterogeneity correlates, albeit imperfectly, with different levels of recombination activity of the mutant RAG proteins. Furthermore, studies in patients and in newly developed animal models carrying hypomorphic RAG mutations have disclosed various mechanisms underlying immune dysregulation in this condition. Careful annotation of clinical outcome and immune reconstitution in RAG‐deficient patients who have received hematopoietic stem cell transplantation has shown that progress has been made in the treatment of this disease, but new approaches remain to be tested to improve stem cell engraftment and durable immune reconstitution. Finally, initial attempts have been made to treat RAG deficiency with gene therapy.     

T-cell receptor variable (V) gene usage by lymphoid populations in T-cell lymphoma.

A panel of monoclonal antibodies specific for TcR V gene families was used to study TcR V region expression in 28 cases of malignant and reactive T-cell expansions including four cases of mixed cellularity Hodgkin's disease (HD) and five reactive cases. TcR V beta 5 gene products were represented in three cases of lymphoblastic malignancy (V beta 5.1, V beta 5.2) and two cases of peripheral T-cell lymphoma (PTCL) (V beta 5.1). In the PTCL cases, the expanded family was found in the absence of clonal TcR gene rearrangements and in one of these cases with Ig JH and Ck clonal gene rearrangements consistent with the presence of a phenotypically and histologically undetectable clonal B-cell population. In a third PTCL case not investigated for genotype, the TCR V alpha 12 family was overrepresented. Expanded TcR V alpha 2 and V beta 5.1 families were identified in HD and V beta 8 and V beta 5.2/V beta 5.3 families in a reactive lymph node and CD3 and CD8-positive blood lymphocytosis respectively. Further study of PTCL and related entities are needed to establish whether expanded TcR families are common in those cases that fail to exhibit clonal TcR gene rearrangement.     

Preselection TCR repertoire predicts CD4+ and CD8+ T-cell differentiation state

T cells must display diversity regarding both the cell state and T-cell receptor (TCR) repertoire to provide effective immunity against pathogens; however, the generation and evolution of cellular T-cell heterogeneity in the adaptive immune system remains unclear. In the present study, a combination of multiplex PCR and immune repertoire sequencing (IR-seq) was used for a standardized analysis of the TCR β-chain repertoire of CD4⁺ naive, CD4⁺ memory, CD8⁺ naive and CD8⁺ memory T cells. We showed that the T-cell subsets could be distinguished from each another with regard to the TCR β-chain (TCR-β) diversity, CDR3 length distribution and TRBV usage, which could be observed both in the preselection and in the post-selection repertoire. Moreover, the Dβ-Jβ and Vβ-Dβ combination patterns at the initial recombination step, template-independent insertion of nucleotides and inter-subset overlap were consistent between the pre- and post-selection repertoires, with a remarkably positive correlation. Taken together, these results support differentiation of the CD4⁺ and CD8⁺ T-cell subsets prior to thymic selection, and these differences survived both positive and negative selection. In conclusion, these findings provide deeper insight into the generation and evolution of TCR repertoire generation.     

T-cell receptor repertoire of human peripheral CD161hiTRAV1-2+ MAIT cells revealed by next generation sequencing and single cell analysis

Mucosal-associated invariant T (MAIT) cells are a T-cell subset that expresses a conserved TRAV1-2 (Vα7.2) T-cell receptor (TCR) chain and the surface marker CD161. They are involved in the defence against microbes as they recognise small organic molecules of microbial origin that are presented by the non-classical MHC molecule 1 (MR1). MAIT cells express a semi-restricted TCR α chain with TRAV1-2 preferentially linked to TRAJ33, TRAJ12, or TRAJ20 which pairs with a limited set of β chains. To investigate the TCR repertoire of human CD161^hiTRAV1-2⁺ T cells in depth we analysed the α and β chains of this T-cell subset by next generation sequencing. Concomitantly we analysed 132 paired α and β chains from single cells to assess the αβ pairing preferences. We found that the CD161^hiTRAV1-2⁺ TCR repertoire in addition to the typical MAIT TCRs further contains polyclonal elements reminiscent of classical αβ T cells.     

No-shows in T-cell responses are frequent for clones of low T-cell receptor affinity

The magnitude of CD8 T-cell responses against intracellular pathogens is thought to primarily depend on the expansion capacity of naïve T cells, given that their recruitment is considered optimal. In the current issue of the European Journal of Immunology [Eur. J. Immunol. 2023. 53: 000-000], Leube et al. challenge these concepts and show that the recruitment of naïve T-cell clones into primary responses can be far from complete. The failure to efficiently recruit T-cell clones occurs more frequently in case of low-affinity interactions of the T-cell receptor with cognate antigen of the pathogen. Using single-cell fate-mapping in the Lm-OVA model, the authors demonstrate that naïve T-cell clones of low affinity in contrast to those of high affinity often do not expand after pathogen encounter. These low-affinity clones are maintained as naïve CD8 T cells that can robustly respond upon secondary encounter with the same pathogen, in particular when the reencountered pathogen contains modifications resulting in improved recognition. Thus, this study indicates that the regulation of the response size of CD8 T cells is yet more elaborate than anticipated and involves control at the level of recruitment and expansion of naïve CD8 T cells.     

Characterization of T-cell subsets and T-cell receptor subgroups in pigtailed macaques using two- and three-color flow cytometry

In order to characterize macaque T-lymphocyte subsets, we used a chromophore from a dinoflagellate, peridinin chlorophyll A protein (PerCP), which, like fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE), can be excited by a 488-nm laser and emits light at 670 nm without spectral overlap with FITC and PE. Mouse monoclonal antibodies were conjugated with FITC, PE, and PerCP to detect CD4+ and CD8+ cells in macaque peripheral blood lymphocytes (PBL) subsets before and after activation and in nonactivated thymocytes. Resting and activated macaque blood CD4+ T-cells could be clearly delineated into discrete subsets with either CD28, CD45RA, or CD45RO as a second marker and CD26, CD29, CD44, or CD69 as a third marker. CD8+ cells were further subdivided by expression of similar combinations of markers. A subset of CD8+ CD28– T-cells in blood expressed the activation marker CD69, suggesting that they were already activated. Virtually all CD4+CD8+, CD4+CD8–, and CD4–CD8+ macaque thymocytes expressed CD2, CD3, and CD18 and not CD25, CD44, or CD45O, but macaque thymocyte subpopulations did differ in their expression of CD28 and CD29. The expression of T-cell receptor (TCR) subgroups on macaque PBL and thymocytes was analyzed before and after activation with staphylococcal enterotoxins (superantigens). The pattern of T-cell variable-region expression in macaques was similar to that seen in humans, with a high frequency of T cells expressing V8. After superantigen stimulation, only minor changes in TCR V expression were detectable in PBL. A dramatic increase in V8 expression was seen after stimulation of macaque thymus with staphylococcal enterotoxin D (SE-D), a minor increase after toxic shock syndrome toxin 1 (TSST-1) stimulation, and a simultaneous decrease in V6 levels.     

T-cell receptor repertoire of circulating gamma delta T-cells in Takayasu's arteritis

We studied T-cell receptor (TCR) repertoire of circulating gamma delta (gammadelta) T-cells in 20 patients with Takayasu's arteritis (TA), 20 healthy controls (HC), 7 follow up TA patients, and 10 patients with rheumatoid arthritis (RA) and 5 Wegener's granulomatosis (WG) patients as disease controls. Patients with TA (8.1 +/- 5.1%) compared to HC (3.7 +/- 2.1%, P = 0.014), RA (4.8 +/- 0.6%, P = 0.032), and WG (4.2 +/- 0.8%, P = 0.030) as well as active TA compared to inactive TA (13.9 +/- 4.1% vs. 4.9 +/- 1.5%; P < 0.001) had higher number of gammadelta T-cells. The numbers of Vdelta1+ cells were significantly higher in patients with TA (40.0 +/- 20.8%) than HC (13.1 +/- 8.0%; P = 0.001), RA (19.5 +/- 1.8%, P = 0.004), and WG (17.0 +/- 3.9%, P = 0.007). The numbers of gammadelta T-cells normalized in all the 7 patients after 180 days of follow up (13.9 +/- 4.1% vs. 6.9 +/- 2.5%; P = 0.001). We also observed higher number of activated and IFN-gamma producing gammadelta T-cells in active TA. Our data show that gammadelta T-cells particularly those bearing Vdelta1 TCR may have an important role in the immunopathogenesis of TA.     

Real-time PCR method for the quantitative analysis of human T-cell receptor gamma and beta gene rearrangements

Analyzing the status of T-cell receptor (TCR) gene rearrangements has been an essential part of deciphering the stages of thymocyte development, understanding the β vs. γδ lineage decision, and characterizing T-cell leukemias. Methods such as PCR and quantitative Southern blotting provide useful information, but also have significant shortcomings such as lack of quantitation in the case of PCR and technical challenges in the case of Southern blotting. Here we describe a real-time PCR method that overcomes many of these shortcomings. This new method shows comparable results for the fraction of unrearranged TCRγ and TCRβ genes in human thymocytes and peripheral blood T cells as Southern blotting, and has the advantages of being simple to perform, highly quantitative, and requiring nanogram quantities of DNA. We also describe a real-time PCR method to quantitate T-cell receptor excision circles formed during TCRβ rearrangements.     

利用T细胞受体变异β基因谱系分析T细胞急性淋巴细胞白血病病人T细胞克隆性

目的:分析T细胞-急性淋巴细胞白血病(T-ALL)病人的T细胞克隆性。方法:利用RT-PCR方法分析6例T-ALL和10例正常人外周血单个核细胞中24个T细胞受体变异β(TCR Vβ)基因的CDR3长度,PCR产物再进一步进行基因扫描和序列分析。结果:3例病人的某些TCR Vβ亚家族T细胞呈单克隆或寡克隆性增殖,主要为Vβ2、3、6、9、21和24。其它3例及正常人均表现为多克隆性增殖T细胞。结论:部分T-ALL来自于TCR Vβ亚家族克隆性增殖T细胞。该方法有助于临床上检测微小残留病变。     

Profiling the repertoire of T-cell receptor beta-chain variable genes in peripheral blood lymphocytes from subjects who have recovered from acute hepatitis B virus infection

The profile of T-cell receptor beta-chain variable （TRBV） genes usually skews in subjects with virus infection or cancer. The gene melting spectral pattern （GMSP） can be used to determine the profile of the TRBV gene family. To explore the portrait of the TRBV family in peripheral blood lymphocytes from subjects who have recovered from acute hepatitis B virus infection （AHI）, peripheral blood mononuclear cells （PBMCs） were separated and further sorted into CD4^＋ and CD8^＋ T-cell subsets. The molecular features of the TRBV complementary determining region 3 （CDR3） motifs were determined using GMSP analysis. When a GMSP profile showed a single peak, the monoclonally expanded TRBV gene was cloned and sequenced. Skewed expansions of multiple TRBV genes were observed among the CD4^＋ and CD8^＋ T-cell subsets and the PBMCs. The frequency of monoclonally expanded TRBV genes in the CD8^＋ T-cell subset was significantly higher than that of the CD4^＋ T-cell subset and the PBMCs. Compared to other members of the TRBV gene family, TRBV11, BV15 and BV20 were predominantly expressed in the repertoire of peripheral blood lymphocytes in recovered AHI subjects. The relatively conserved amino acid motifs of TRBV5.1 and BV20 CDR3 were also detected in the CD4^＋ and CD8^＋ T-cell subsets. These results demonstrate the presence of multiple biased TRBV families in recovered AHI subjects. TRBV11, BV15 and BV20, especially from the CD8＋ T-cell subset, may be relevant to the pathogenesis of subjects with AHh The preferentially selected TRBV5.1 and BV20 with the relatively conserved CDR3 motif may be potential targets for personalized treatments of chronic HBV infection.     

Omenn's syndrome occurring in patients without mutations in recombination activating genes

Omenn syndrome (OS) is characterised by hepatosplenomegaly, lymphadenopathy, erythema, eosinophilia, elevated IgE, oligoclonal T cell expansions and recombinase activating gene (RAG) mutations. We investigated 9 cases of OS to correlate genotype with immunophenotype using a two-color flow cytometry with monoclonal antibodies against CD3 and TCRVB families to map TCRVB usage. T and B clonal cell populations were examined in peripheral blood lymphocytes by PCR and sequencing of TCRB/TCRG T cell and IGH FR2/FR3 B cell products. RAG and Artemis genes were sequenced from genomic DNA. All patients demonstrated absent TCRVB families; six had predominant TCRVB families, six oligoclonal TCR gene rearrangements including TCRGD rearrangements. One demonstrated functional IGH rearrangement, an observation not previously reported. In this clinically homogeneous population, with similar immunological phenotype, RAG mutations were identified in only 2/9 patients. OS is a genetically heterogeneous condition, and patients with similar immunophenotypes may have as yet unidentified gene defects.     

Demonstration of clonality in T-cell lymphoma using an anti-T-cell receptor variable region antibody panel

Frozen sections from 35 T-cell lymphomas were stained with the Diversi-T alpha beta T-Cell Receptor panel which includes seven antibodies to T-cell receptor variable region gene products. In five cases a monoclonal population of T-cells could be demonstrated (one case V beta 5+, three cases V beta 8+ and one case V beta 6+) and in a further case a biclonal population (V beta 5+ and V beta 8+). We conclude that this antibody panel is of limited usefulness for the demonstration of clonality in T-cell lymphoma.     

Diagnostic and prognostic significance of clonal T-cell receptor beta gene rearrangements in lymph nodes of patients with mycosis fungoides

In this study, 25 involved and uninvolved lymph nodes from 22 patients with mycosis fungoides (MF) and seven dermatopathic lymph nodes from patients with benign skin disorders were studied for the presence of clonal T-cell receptor beta (TCRβ) gene rearrangements by Southern blot analysis. These results were correlated with the histological classification, follow-up data, and survival. The results of the histological classification and Southern blot analysis were concordant in 26 of 32 cases. Clonal TCRβ gene rearrangements were found in all six MF lymph nodes showing (partial) effacement of the normal lymph node architecture, but in none of the eight uninvolved dermatopathic MF lymph nodes and in none of the seven dermatopathic control lymph nodes. In addition, in 5 of 11 dermatopathic MF lymph nodes that were considered to have early involvement by MF at histological examination, clonal TCRβ gene rearrangements were detected. In the group of MF patients with dermatopathic lymphadenopathy, patients with detectable clonal T-cell populations had a significantly shorter survival than patients without such a population (P<0.01). The results of this study indicate that within the group of dermatopathic MF lymph nodes, prognostically different groups can be distinguished and that TCRβ gene rearrangement analysis may be an important adjunct in the early diagnosis of lymph node involvement by MF.     

B‐cell receptor repertoire sequencing in patients with primary immunodeficiency: a review

The advent of next‐generation sequencing (NGS) now allows a detailed assessment of the adaptive immune system in health and disease. In particular, high‐throughput B‐cell receptor (BCR) repertoire sequencing provides detailed information about the functionality and abnormalities of the B‐cell system. However, it is mostly unknown how the BCR repertoire is altered in the context of primary immunodeficiencies (PID) and whether findings are consistent throughout phenotypes and genotypes. We have performed an extensive literature search of the published work on BCR repertoire sequencing in PID patients, including several forms of predominantly antibody disorders and combined immunodeficiencies. It is somewhat surprising that BCR repertoires, even from severe clinical phenotypes, often show only mild abnormalities and that diversity or immunoglobulin gene segment usage is generally preserved to some extent. Despite the great variety of wet laboratory and analytical methods that were used in the different studies, several findings are common to most investigated PIDs, such as the increased usage of gene segments that are associated with self‐reactivity. These findings suggest that BCR repertoire characteristics may be used to assess the functionality of the B‐cell compartment irrespective of the underlying defect. With the use of NGS approaches, there is now the opportunity to apply BCR repertoire sequencing to multiple patients and explore the PID BCR repertoire in more detail. Ultimately, using BCR repertoire sequencing in translational research could aid the management of PID patients by improving diagnosis, estimating functionality of the immune system and improving assessment of prognosis.     

The role of self-recognition in receptor repertoire development

The role of self-antigen recognition in the development of T and B cells of the adaptive immune system has been studied in several different ways. We have shown that CD4 T cells are selected on selfpeptide:self-MHC class II ligands, and in the periphery, they are sustained by contact with the same or similar ligands. We have also observed that B cells are positively selected on unknown and presumed self-ligands. We have used this information to explore autoimmune diseases as well. Finally, we have recently identified the innate immune system as playing a crucial role in regulating expression of costimulatory molecules that are required for induction of adaptive immune responses. People whose work is included in this review are as follows: Chuck Annicelli, Joanne Appicelli, Avlin Barlow, Jody Baron, Michael Carrithers, Chaoqun Chen, Alexander Chervonsky, Beth Cohen, Bonnie Dittel, Tori Evans, Jennifer Granata, Fridrika Hardardottir, Xin He, Jon Kaye, Matthew Levine. Yang Liu, Grigori Loysev, Ruslan Medzhitov, Paula Preston-Hurlburt, Eve Robinson, Alexander Rudensky, Derek Sant’Angelo, John Shanabrough, Christophe Viret, Irene Visintin, and Sue Wong     

CXCR3-mediated T-cell chemotaxis involves ZAP-70 and is regulated by signalling through the T-cell receptor

The chemokine receptor CXCR3 is critical for the function of activated T cells. We studied the molecular mechanisms of CXCR3 signalling. The addition of CXCR3 ligands to normal human T cells expressing CXCR3 led to the tyrosine phosphorylation of multiple proteins. Addition of the same ligands to Jurkat T cells engineered to express CXCR3 induced tyrosine phosphorylation of proteins with molecular weights similar to those in normal cells. Immunoblotting with phosphotyrosine-specific antibodies identified Zeta-associated protein of 70,000 molecular weight (ZAP-70), linker for the activation of T cells (LAT), and phospholipase-C-gamma1 (PLCgamma1) to be among the proteins that become phosphorylated upon CXCR3 activation. ZAP-70 was phosphorylated on tyrosine 319, LAT on tyrosines 171 and 191, and PLCgamma1 on tyrosine 783. The ZAP-70 inhibitor piceatannol reduced CXCR3-mediated tyrosine phosphorylation of ZAP-70, LAT, PLCgamma1 and mitogen-activated protein kinase Erk and it reduced CXCL10-mediated chemotaxis of both CXCR3-transfected Jurkat T cells and normal T cells expressing CXCR3. These results are consistent with the involvement of ZAP-70 in CXCR3-mediated protein tyrosine phosphorylation and CXCR3-induced T-cell chemotaxis. Studies with the Lck-deficient Jurkat T-cell line, JCAM1.6, demonstrated that phosphorylation of ZAP-70 after CXCR3 activation is a Lck-dependent process. Finally, stimulating CXCR3-expressing Jurkat T cells and normal T cells expressing CXCR3 through the T-cell receptor attenuated CXCR3-induced tyrosine phosphorylation and CXCR3-mediated T-cell migration, indicating the occurrence of cross-talk between T-cell receptor and CXCR3-signalling pathways. These results shed light on the mechanisms of CXCR3 signalling. Such information could be useful when designing therapeutic strategies to regulate T-cell function.     

Repertoire of the αβ T-cell receptor in the intestine

Summary:  The majority of T cells in the human and mouse intestine express the T-cell receptor (TCR) as an αβ heterodimer on their cell surface. As the major recognition element of antigens in the context of major histocompatibility complex-derived proteins, an examination of the structure of the αβTCR in intestines has provided significant insights into the potential function of these cells and the major determinants that drive their selection. Studies in the human intestine have shown that the repertoires of intraepithelial lymphocytes (IELs), and likely lamina propria lymphocytes, are polyclonal before and shortly after birth, with the repertoire becoming oligoclonal in adults. Similarly, in adult mice the repertoire is oligoclonal, while in the newborn it is polyclonal. Investigations in mice have shown that some T cells may evade thymic selection. The population size and oligoclonality of IELs is influenced by the microbial content of the luminal microenvironment. This microenvironment probably directly determines the TCR repertoire. Studies in human inflammatory bowel disease (IBD) indicate that inflammation further skews the TCR repertoire. We speculate that dominant antigens associated with the pathogenesis of IBD are responsible for such skewing and that identifying the antigenic drivers may shed light on the environmental factors that trigger or potentiate human IBD.     

Regulation of human T-cell homing receptor expression in cutaneous bacterial infection

We investigated the regulation of T-cell homing receptors in infectious disease by evaluating the cutaneous lymphocyte antigen (CLA) in human leprosy. We found that CLA-positive cells were enriched in the infectious lesions associated with restricting the growth of the pathogen Mycobacterium leprae, as assessed by the clinical course of infection. Moreover, CLA expression on T cells isolated from the peripheral blood of antigen-responsive tuberculoid leprosy patients increased in the presence of M. leprae (2.4-fold median increase; range 0.8-6.1, n = 17), but not in unresponsive lepromatous leprosy patients (1.0-fold median increase; range 0.1-2.2, n = 10; P < 0.005). Mycobacterium leprae specifically up-regulated the skin homing receptor, CLA, but not alpha(4)/beta(7), the intestinal homing receptor, which decreased on T cells of patients with tuberculoid leprosy after antigen stimulation (2.2-fold median decrease; range 1.6-3.4, n = 3). Our data indicate that CLA expression is regulated during the course of leprosy infection and suggest that T-cell responsiveness to a microbial antigen directs antigen-specific T cells to the site of infection.     

T-cell antigen receptor beta-chain variable region families: a study of their distribution in normal and reactive tissues

The beta chain of the T-cell antigen receptor is constructed using a variable region gene from one of approximately 20 variable gene families. Antibodies that react with the products of these gene families have been used to demonstrate clonal proliferation of T cells. Here, we describe the expression of two beta-chain variable region families in normal and reactive lymphoid tissues. In all the tissues studied, expression was scattered throughout the T-cell areas, without any clustering of expression.