Expansion of clonotype-restricted HLA-identical maternal CD4+ T cells in a patient with severe combined immunodeficiency and a homozygous mutation in the Artemis gene

We have observed a male infant with severe combined immunodeficiency (SCID) responsible for Artemis gene mutation, in whom marked expansion of the transplacentally grafted maternal CD4(+) T cells was observed in various tissues. His class I and II major histocompatibility antigens (MHC) were identical to his mother's. We analyzed the T-cell populations within target tissues at a molecular level in order to determine whether different T-cell clonotypes are expanded in different types of tissue. Prior to T-cell expansion, the T-cell receptor variable beta (TCRBV) 5.1 subfamily predominated in peripheral blood (PB) lymphocytes. Third complementarity determining region (CDR3) size spectratyping and amino acid sequencing showed that the range of T-cell clonotypes was very restricted. After T-cell expansion, different TCRBV subfamilies were found to predominate in different target tissues; these included TCRBV 5.1 and 17 in the PB, TCRBV 13 and 21.3 in the bone marrow, and TCRBV 17 in lymph nodes. CDR3 size analysis showed that the expression of different proliferating T-cell clonotypes remained restricted after T-cell expansion. These results indicate that highly restricted maternal T-cell clonotypes can markedly expand, possibly in response to tissue-specific antigens, in a MHC-identical recipient.     

Detection of RAG mutations and prenatal diagnosis in families presenting with either T-B- severe combined immunodeficiency or Omenn's syndrome

It has been recently shown that mutations in both of the recombination activating genes RAG1 and RAG2 are involved in each of the two different types of severe combined immunodeficiency (SCID) syndromes: T-B- SCID and Omenn's syndrome (OS). The objective of the study was to search for novel mutations in the RAG genes and to offer prenatal diagnosis for families that have been identified as at risk of T-B- SCID or OS. Mutation analyses of polymerase chain reaction products of RAG1/RAG2 genes were performed in 14 cases (T-B- SCID = 6 and OS = 8). Consanguinity was reported in seven (50%) families. Four missense mutations in the RAG2 gene in six of eight OS patients and in four of six T-B- SCID patients were detected. The C1845T transition leading to a Tre215Ile substitution is a novel mutation. All but one of the patients were homozygous for the detected mutations, possibly reflecting the consanguinity in these families and the relative rarity of the disease-causing mutations. In addition, three putative polymorphic sites were found. Prenatal diagnosis was offered to seven families, but three of them declined genetic counseling for religious reasons. In the remaining families, four pregnancies were successfully completed, and in one case, the family chose to have an abortion because of a homozygous mutation. Mutations in RAG1/RAG2 genes were detected in only some of the T-B- SCID or OS patients, and the molecular basis for the remaining cases has yet to be elucidated. Important factors such as religious beliefs need to be considered when offering prenatal diagnosis to certain families.     

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.     

Immunological characteristics and T‐cell receptor clonal diversity in children with systemic juvenile idiopathic arthritis undergoing T‐cell‐depleted autologous stem cell transplantation

Children with systemic Juvenile Idiopathic Arthritis (sJIA), the most severe subtype of JIA, are at risk from destructive polyarthritis and growth failure, and corticosteroids as part of conventional treatment can result in osteoporosis and growth delay. In children where there is failure or toxicity from drug therapies, disease has been successfully controlled by T‐cell‐depleted autologous stem cell transplantation (ASCT). At present, the immunological basis underlying remission after ASCT is unknown. Immune reconstitution of T cells, B cells, natural killer cells, natural killer T cells and monocytes, in parallel with T‐cell receptor (TCR) diversity by analysis of the β variable region (TCRVb) complementarity determining region‐3 (CDR3) using spectratyping and sequencing, were studied in five children with sJIA before and after ASCT. At time of follow up (mean 11·5 years), four patients remain in complete remission, while one child relapsed within 1 month of transplant. The CD8⁺ TCRVb repertoire was highly oligoclonal early in immune reconstitution and re‐emergence of pre‐transplant TCRVb CDR3 dominant peaks was observed after transplant in certain TCRVb families. Further, re‐emergence of pre‐ASCT clonal sequences in addition to new sequences was identified after transplant. These results suggest that a chimeric TCR repertoire, comprising T‐cell clones developed before and after transplant, can be associated with clinical remission from severe arthritis.     

Newborn screening for severe combined immunodeficiency and T‐cell lymphopenia

The development of a T cell receptor excision circle (TREC) assay utilizing dried blood spots (DBS) made possible universal newborn screening (NBS) for severe combined immunodeficiency (SCID) as a public health measure. Upon being flagged by an abnormal screening test in a SCID screening program, an infant can receive further diagnostic testing for SCID in the neonatal period, prior to onset of infectious complications, to permit immediate institution of protective measures and definitive, life‐saving treatment to establish a functional immune system. SCID screening is now the accepted standard of care in state public health departments across the United States, and it is being adopted in many countries. It has proven effective, with infants having this otherwise inapparent but serious, rare disorder achieving survival and immune reconstitution. In addition to bringing to attention infants with the primary screening target diseases, typical SCID and leaky SCID (due to hypomorphic mutations in known SCID genes), the NBS assay for insufficient TRECs in DBS also reveals infants with non‐SCID T lymphopenic conditions. Experience has accumulated regarding the range and limitations of diagnoses of newborns with low TRECs and low T cells. Previously unknown immune defects have been discovered, as well as conditions not formerly recognized to have low T cells in the neonatal period.     

Analysis of mutations and recombination activity in RAG-deficient patients

Mutations in the recombination activating genes (RAG1 or RAG2) can lead to a variety of immunodeficiencies. Herein, we report 5 cases of RAG deficiency from 5 families: 3 of Omenn syndrome, 1 of severe combined immunodeficiency, and 1 of combined immunodeficiency with oligoclonal TCRγδ(+) T cells, autoimmunity and cytomegalovirus infection. The genetic defects were heterogeneous and included 6 novel RAG mutations. All missense mutations except for Met443Ile in RAG2 were located in active core regions of RAG1 or RAG2. V(D)J recombination activity of each mutant was variable, ranging from half of the wild type activity to none, however, a significant decrease in average recombination activity was demonstrated in each patient. The reduced recombination activity of Met443Ile in RAG2 may suggest a crucial role of the non-core region of RAG2 in V(D)J recombination. These findings suggest that functional evaluation together with molecular analysis contributes to our broader understanding of RAG deficiency.     

Clonotypic analysis of T cell reconstitution after haematopoietic stem cell transplantation (HSCT) in patients with severe combined immunodeficiency

Haematopoietic stem cell transplantation (HSCT) is performed for treatment of a broad spectrum of illnesses. Reconstitution of an intact immune system is crucial after transplantation to avoid infectious complications, and above all, the establishment of T cell receptor (TCR) diversity is the most important goal in the procedure. Until recently, little has been known of the mechanism of T cell reconstitution in the very early period after HSCT. In this study, we analysed TCR repertoires sequentially in four patients with severe combined immunodeficiency (SCID) before and after HSCT. In all patients, the TCR repertoires were extremely abnormal before HSCT, whereas after transplantation there was progressive improvement in TCR diversity, based on analysis of the TCR Vbeta repertoire and CDR3 size distributions. Somewhat unexpectedly, there was a significant but transient expansion of TCR diversity 1 month after transplantation in all cases. Clonotypic analysis of TCRs performed in one case showed that many T cell clones shared identical CDR3 sequences at 1 month and that the shared fraction decreased progressively. These results indicate that early expansion of TCR diversity may reflect transient expansion of pre-existing mature T cells from the donor blood, independent of de novo T cell maturation through the thymus.     

Unusual clinical and immunologic manifestations of transplacentally acquired maternal T cells in severe combined immunodeficiency

The persistence of transplacentally transferred maternal T cells is common in infants with severe combined immunodeficiency (SCID), occurring in more than half of patients with SCID undergoing transplantation at our institution. These T cells respond poorly to mitogens in vitro but can cause cutaneous graft-versus-host disease; however, other effects of these cells are unknown. We describe 2 infants with SCID who had unusual problems associated with transplacentally transferred maternal T cells. Patient 1 was a 5-month-old girl with Janus kinase 3-deficient SCID who had 4% circulating CD3(+) T cells but no lymphocyte proliferative response to mitogens. Although the number of T cells increased after 2 nonchemoablated, T cell-depleted, haploidentical, paternal bone marrow transplantations, T-cell function failed to develop, and she became pancytopenic. Restriction fragment length polymorphism studies of flow cytometry-sorted blood T cells revealed all to be of maternal origin. A subsequent nonchemoablated, T cell-depleted maternal transplantation resulted in normal T-cell function and marrow recovery. Patient 2 was a 9-month-old girl with IL-7Ralpha-deficient SCID who presented with autoimmune pancytopenia. She had 8% blood T cells (all CD45RO(+)) but no response to mitogens. High-resolution HLA sequence-specific priming typing detected both maternal haplotypes, indicating the presence of maternal cells. Her pancytopenia resolved after treatment with rituximab and was thought to be due to host B-cell activation by transplacentally acquired maternal T cells. Persistent transplacentally acquired maternal T cells in infants with SCID can mediate immunologic functions despite failing to respond to mitogens in vitro. We present evidence that these cells can cause allograft rejection and immune cytopenias.     

T‐bet regulates differentiation of forkhead box protein 3+ regulatory T cells in programmed cell death‐1‐deficient mice

Programmed cell death‐1 (PD‐1) plays an important role in peripheral T cell tolerance, but whether or not it affects the differentiation of helper T cell subsets remains elusive. Here we describe the importance of PD‐1 in the control of T helper type 1 (Th1) cell activation and development of forkhead box protein 3 (FoxP3⁺) regulatory T cells (T_regs). PD‐1‐deficient T cell‐specific T‐bet transgenic (P/T) mice showed growth retardation, and the majority died within 10 weeks. P/T mice showed T‐bet over‐expression, increased interferon (IFN)‐γ production by CD4⁺ T cells and significantly low FoxP3⁺ T_reg cell percentage. P/T mice developed systemic inflammation, which was probably induced by augmented Th1 response and low FoxP3⁺ T_reg count. The study identified a unique, previously undescribed role for PD‐1 in Th1 and T_reg differentiation, with potential implication in the development of Th1 cell‐targeted therapy.     

Evidence for a preferential Vβ usage by the T cells which adoptively transfer diabetes in NOD mice

Non-obese diabetic (NOD) mice become spontaneously diabetic as a result of a genetically programmed autoimmune process mediated by autoreactive T lymphocytes and directed against β cell antigen(s). Studies dealing with T cell receptor (TcR) variable (V) gene usage by such autoreactive T lymphocytes have given contrasted results. Various reasons may explain these discrepancies: the multiplicity of antigenic epitopes putatively recognized by T cells, the ambiguity between specifically committed T cells and passenger lymphocytes homing randomly to the pancreas, the necessarily limited size of the T cell clone panels which have been analyzed for TcR rearrangements and, last but not least, the flexibility of T cell repertoires. To circumvent some of these difficulties, we have decided to concentrate upon the T cell population present in diseased animals and capable of transferring diabetes into young naive NOD recipients. This population, composed of CD4⁺ and CD8⁺ T cells, is presumably committed against the relevant β cell antigens and is the most likely to reveal a bias in V gene usage if such a bias does indeed exist. To find out whether certain V_β genes are more frequently used than others by such pathogenic T cells, T lymphocytes from diabetic donors have been depleted in vitro of defined V_β subsets before being reinoculated into permissive recipients. Out of four V_β families probed under such conditions, three (V_β8, V_β5 and V_β11 are neutral. Their absence neither increases nor reduces the final incidence of successful transfers, indicating that these gene segments are not preferentially used. In contrast, the depletion of V_β6-positiveT cells results in a severe reduction of transfers, suggesting that V_β6 gene is used with a relatively high frequency by diabetogenic CD4⁺ and/or CD8⁺ T cells. To define more precisely which subset uses V_β6 gene preferentially, we have performed mixing experiments with deleted and intact subsets. The results, based on disease transfer and insulitis severity, indicate that the V_β6 bias affects predominantly the CD4⁺ subset. Thus, at variance with several studies concluding that V gene usage in NOD mice is heterogeneous, our present data suggest that disease transferring T cells use a relatively restricted set of V_β genes.     

T cell receptor Vbeta repertoire of T lymphocytes and T regulatory cells by flow cytometric analysis in healthy children

Evaluation of the T cell receptor (TCR) Vbeta repertoire by flow cytometric analysis has been used for studying the T cell compartments for diseases in which T cells are implicated in the pathogenesis. For the interpretation of these studies information is needed about Vbeta usage in healthy individuals and there are few data for normal usage in paediatric populations. We examined the T lymphocyte (sub)populations in 47 healthy controls (age range: 3 months-16 years). We found non-random Vbeta usage with skewed reactivity of some families towards CD4+ or CD4- T cells. Importantly, there appeared to be no significant change in Vbeta usage according to age group. Some controls showed expansions in some Vbeta families, although incidence of such expansions was low. We went on to examine the repertoire of CD4+CD25(Bright) T regulatory cells in 25 healthy controls. We found overlapping quantitative usage for each of the Vbeta families between CD4+CD25- and CD4+CD25(Bright) T cells. However, there was a significant preferential usage for five Vbeta families and decreased usage of two Vbeta families in the CD4+CD25(Bright) T cells, suggesting that although they overlap there may be subtle but important differences in the TCR repertoire of T regulatory cells.     

Lack of selective Vβ deletion in peripheral CD4+ T cells of human immunodeficiency virus-infected infants

To investigate the possibility of superantigen-mediated deletions of T cells expressing particular T cell receptor Vβ (TcR Vβ) gene segments during human immunodeficiency virus (HIV) infection, TcR Vp usage in CD4⁺ and CD8⁺ subsets was analyzed in a cohort of infants maternally infected by HIV and in a group of healthy neonates. We used a semi-quantitative anchored polymerase chain reaction technique together with cytofluorographic analysis with anti-Vβ monoclonal antibodies. The representation of the 24 vβ families in CD4⁺ and CD8⁺ T cells from normal neonates was very similar to that in adults. Preferential expression of Vβ2 in the CD4⁺ subset was observed in both the neonates and in healthy adults. The representation of the 24 Vβ families in peripheral CD4⁺ T cells from the HIV-infected infants showed no selective vβ deletion, even when the CD4⁺ subset was globally depleted. Moreover, the main characteristics of the control group (predominance of certain Vβ families and Vβ2 skewing towards the CD4⁺ subset) were also present in all the HIV-infected infants.     

CD8+ CD122+ regulatory T cells contain clonally expanded cells with identical CDR3 sequences of the T‐cell receptor β‐chain

We identified CD8⁺ CD122⁺ regulatory T cells (CD8⁺ CD122⁺ Treg cells) and reported their importance in maintaining immune homeostasis. The absence of CD8⁺ CD122⁺ Treg cells has been shown to lead to severe systemic autoimmunity in several mouse models, including inflammatory bowel diseases and experimental autoimmune encephalomyelitis. The T‐cell receptors (TCRs) expressed on CD8⁺ CD122⁺ Treg cells recognize the target cells to be regulated. To aid in the identification of the target antigen(s) recognized by TCRs of CD8⁺ CD122⁺ Treg cells, we compared the TCR diversity of CD8⁺ CD122⁺ T cells with that of conventional, naive T cells in mice. We analysed the use of TCR‐Vβ in the interleukin 10‐producing population of CD8⁺ CD122⁺ T cells marked by high levels of CD49d expression, and found the significantly increased use of Vβ13 in these cells. Immunoscope analysis of the complementarity‐determining region 3 (CDR3) of the TCR β‐chain revealed remarkable skewing in a pair of Vβ regions, suggesting the existence of clonally expanded cells in CD8⁺ CD122⁺ T cells. Clonal expansion in Vβ13⁺ cells was confirmed by determining the DNA sequences of the CDR3s. The characteristic TCR found in this study is an important building block for further studies to identify the target antigen recognized by CD8⁺ CD122⁺ Treg cells.     

Frequent occurrence of high affinity T cells against MELOE‐1 makes this antigen an attractive target for melanoma immunotherapy

We recently showed that the infusion of tumor infiltrating lymphocytes specific for the MELOE‐1 antigen was associated with a prolonged relapse‐free survival for HLA‐A2⁺ melanoma patients who received tumor infiltrating lymphocytes therapy. Here, we characterized the MELOE‐1/A2‐specific T‐cell repertoire in healthy donors and melanoma patients to further support an immunotherapy targeting this epitope. Using tetramer enrichment followed by multicolor staining, we found that MELOE‐1‐specific T cells were present in the blood of healthy donors and patients at similar frequencies (around 1 in 1×10⁵ CD8⁺ cells). These cells mainly displayed a naïve phenotype in 4/6 healthy donors and 3/6 patients, whereas high proportions of memory cells were observed in the remaining individuals of both groups. There was a recurrent usage of the Vα12.1 chain for 17/18 MELOE‐1‐specific T‐cell clones derived from healthy donors or patients, associated with diverse Vβ chains and V(D)J junctional sequences. All clones derived from melanoma patients (9/9) were reactive against the MELOE‐1_36–44 peptide and against HLA‐A2⁺ melanoma cell lines. This study documents the existence of a large TCR repertoire specific for the MELOE‐1/A2 epitope and its capacity to give rise to antitumor CTL that supports the development of immunotherapies targeting this epitope.     

Predominant rearrangements of the T cell receptor β-chain gene in blood lymphocyte populations stimulated with autologous tumor cells suggest clonal T cell expansion in two of fourteen cases

T cell responses against autologous tumors with samples from patients with a variety of tumors were examined. The abilities of T lymphocytes to lyse the autologous tumor cells were analyzed after short-term mixed lymphocyte/tumor cell cultures (MLTC). Southern blot analysis was used to evaluate whether particular rearrangements of the TCR β-chain gene predominate in these cultures. Tumor specific lysis could be induced in a proportion of the mixed cultures. In two cases enrichment of T lymphocytes with similar TCR β-chain gene rearrangements was detected after repeated stimulations with autologous tumor cells.     

Patients with T+/low NK+ IL‐2 receptor γ chain deficiency have differentially‐impaired cytokine signaling resulting in severe combined immunodeficiency

X‐linked severe combined immunodeficiency (X‐SCID) leads to a T^?NK^?B⁺ immunophenotype and is caused by mutations in the gene encoding the IL‐2 receptor γ‐chain (IL2RG). IL2RG^R222C leads to atypical SCID with a severe early onset phenotype despite largely normal NK‐ and T‐cell numbers. To address this discrepancy, we performed a detailed analysis of T, B, and NK cells, including quantitative STAT phosphorylation and functional responses to the cytokines IL‐2, IL‐4, IL‐15, and IL‐21 in a patient with the IL2RG^R222C mutation. Moreover, we identified nine additional unpublished patients with the same mutations, all with a full SCID phenotype, and confirmed selected immunological observations. T‐cell development was variably affected, but led to borderline T‐cell receptor excision circle (TREC) levels and a normal repertoire. T cells showed moderately reduced proliferation, failing enhancement by IL‐2. While NK‐cell development was normal, IL‐2 enhancement of NK‐cell degranulation and IL‐15‐induced cytokine production were absent. IL‐2 or IL‐21 failed to enhance B‐cell proliferation and plasmablast differentiation. These functional alterations were reflected by a differential impact of IL2RG^R222C on cytokine signal transduction, with a gradient IL‐4R222C causes a consistently severe clinical phenotype that is not predicted by the variable and moderate impairment of T‐cell immunity or TREC analysis.     

Vβ gene repertoires in T cells expanded in local self-healing and lethal systemic murine cutaneous leishmaniasis

Inbred mice infected with Leishmania major promastigotes display two different courses of leishmaniasis: resistant strains develop self-healing local sores, while susceptible strains show progressive systemic disease with lethal outcome. Resistance predominantly correlates with the production of T helper type 1 (TH1) lymphokines and susceptibility with production of TH2-type lymphokines. Here, we analyzed whether this TH phenotype difference correlates with expression of particular T cell receptor Vβ chains. Our results show that T cells expand strongly during infection in all groups of mice and invariantly express the same Vβ gene families as prior to infection. Our data indicate that TH1 and TH2 cells use similar Vβ gene families, and argue against the engagement of a restricted set of Vβ by dominant determinants associated with L. major.