A novel immunodeficiency associated with hypomorphic RAG1 mutations and CMV infection

Amorphic mutations in the recombination activating genes RAG1 and RAG2 have been reported to cause T- B- SCID, whereas hypomorphic mutations led to the expansion of a few autoimmune T cell clones responsible for the Omenn syndrome phenotype. We report here a novel clinical and immunological phenotype associated with recessive RAG1 hypomorphic mutations in 4 patients from 4 different families. The immunological phenotype consists of the oligoclonal expansion of TCR gammadelta T cells combined with TCR alphabeta T cell lymphopenia. The clinical phenotype consists of severe, disseminated CMV infection and autoimmune blood cell manifestations. Repertoire studies suggest that CMV infection, in the setting of this particular T cell immunodeficiency, may have driven the TCR gammadelta T cell clonal expansion. This observation extends the range of clinical and immunological phenotypes associated with RAG mutations, emphasizing the role of the genetic background and microbial environment in determining disease phenotype.     

A new type of radiosensitive T-B-NK+ severe combined immunodeficiency caused by a LIG4 mutation

V(D)J recombination of Ig and TCR loci is a stepwise process during which site-specific DNA double-strand breaks (DSBs) are made by RAG1/RAG2, followed by DSB repair by nonhomologous end joining. Defects in V(D)J recombination result in SCID characterized by absence of mature B and T cells. A subset of T-B-NK+ SCID patients is sensitive to ionizing radiation, and the majority of these patients have mutations in Artemis. We present a patient with a new type of radiosensitive T-B-NK+ SCID with a defect in DNA ligase IV (LIG4). To date, LIG4 mutations have only been described in a radiosensitive leukemia patient and in 4 patients with a designated LIG4 syndrome, which is associated with chromosomal instability, pancytopenia, and developmental and growth delay. The patient described here shows that a LIG4 mutation can also cause T-B-NK+ SCID without developmental defects. The LIG4-deficient SCID patient had an incomplete but severe block in precursor B cell differentiation, resulting in extremely low levels of blood B cells. The residual D(H)-J(H) junctions showed extensive nucleotide deletions, apparently caused by prolonged exonuclease activity during the delayed D(H)-J(H) ligation process. In conclusion, different LIG4 mutations can result in either a developmental defect with minor immunological abnormalities or a SCID picture with normal development.     

Reconstitution of T cell receptor signaling in ZAP-70-deficient cells by retroviral transduction of the ZAP-70 gene

A variant of severe combined immunodeficiency syndrome (SCID) with a selective inability to produce CD8 single positive T cells and a signal transduction defect in peripheral CD4+ cells has recently been shown to be the result of mutations in the ZAP-70 gene. T cell receptor (TCR) signaling requires the association of the ZAP-70 protein tyrosine kinase with the TCR complex. Human T cell leukemia virus type I- transformed CD4+ T cell lines were established from ZAP-70-deficient patients and normal controls. ZAP-70 was expressed and appropriately phosphorylated in normal T cell lines after TCR engagement, but was not detected in T cell lines from ZAP-70-deficient patients. To determine whether signaling could be reconstituted, wild-type ZAP-70 was introduced into deficient cells with a ZAP-70 retroviral vector. High titer producer clones expressing ZAP-70 were generated in the Gibbon ape leukemia virus packaging line PG13. After transduction, ZAP-70 was detected at levels equivalent to those observed in normal cells, and was appropriately phosphorylated on tyrosine after receptor engagement. The kinase activity of ZAP-70 in the reconstituted cells was also appropriately upregulated by receptor aggregation. Moreover, normal and transduced cells, but not ZAP-70-deficient cells, were able to mobilize calcium after receptor ligation, indicating that proximal TCR signaling was reconstituted. These results indicate that this form of SCID may be corrected by gene therapy.     

肝脾γδT细胞淋巴瘤一例

肝脾γδT细胞淋巴瘤是一种罕见的高度恶性的外周T细胞淋巴瘤，表达T细胞标志CD2、CD3以及γδ型T细胞受体。病理表现为肿瘤性T细胞在肝脏血窦和脾脏红髓的弥漫性浸润，不形成结节，易造成误诊。通过对患者肝脏活检标本、脾脏穿刺标本和骨髓标本的免疫表型及TCR受体分析，诊断为1例成人肝脾γδT细胞淋巴瘤。结合文献复习，可以认为免疫表型及TCR受体分析是诊断肝脾γδT细胞淋巴瘤的必要手段。     

Analysis of mutations from SCID and Omenn syndrome patients reveals the central role of the Rag2 PHD domain in regulating V(D)J recombination

Rag2 plays an essential role in the generation of antigen receptors. Mutations that impair Rag2 function can lead to severe combined immunodeficiency (SCID), a condition characterized by complete absence of T and B cells, or Omenn syndrome (OS), a form of SCID characterized by the virtual absence of B cells and the presence of oligoclonal autoreactive T cells. Here, we present a comparative study of a panel of mutations that were identified in the noncanonical plant homeodomain (PHD) of Rag2 in patients with SCID or OS. We show that PHD mutant mouse Rag2 proteins that correspond to those found in these patients greatly impaired endogenous recombination of Ig gene segments in a Rag2-deficient pro-B cell line and that this correlated with decreased protein stability, impaired nuclear localization, and/or loss of the interaction between Rag2 and core histones. Our results demonstrate that point mutations in the PHD of Rag2 compromise the functionality of the entire protein, thus explaining why the phenotype of cells expressing PHD point mutants differs from those expressing core Rag2 protein that lacks the entire C-terminal region and is therefore devoid of the regulation imposed by the PHD. Together, our findings reveal the various deleterious effects of PHD Rag2 mutations and demonstrate the crucial role of this domain in regulating antigen receptor gene assembly. We believe these results reveal new mechanisms of immunodeficiency in SCID and OS.     

Shared reactivity of V{delta}2(neg) {gamma}{delta} T cells against cytomegalovirus-infected cells and tumor intestinal epithelial cells

Long-lasting expansion of Vdelta2(neg) gammadelta T cells is a hallmark of cytomegalovirus (CMV) infection in kidney transplant recipients. The ligands of these cells and their role remain elusive. To better understand their immune function, we generated gammadelta T cell clones from several transplanted patients. Numerous patient Vdelta1(+), Vdelta3(+), and Vdelta5(+) gammadelta T cell clones expressing diverse Vgamma chains, but not control Vgamma9Vdelta2(+) T clones, displayed strong reactivity against CMV-infected cells, as shown by their production of tumor necrosis factor-alpha. Vdelta2(neg) gammadelta T lymphocytes could also kill CMV-infected targets and limit CMV propagation in vitro. Their anti-CMV reactivity was specific for this virus among herpesviridae and required T cell receptor engagement, but did not involve major histocompatibility complex class I molecules or NKG2D. Vdelta2(neg) gammadelta T lymphocytes expressed receptors essential for intestinal homing and were strongly activated by intestinal tumor, but not normal, epithelial cell lines. High frequencies of CMV- and tumor-specific Vdelta2(neg) gammadelta T lymphocytes were found among patients' gammadelta T cells. In conclusion, Vdelta2(neg) gammadelta T cells may play a role in protecting against CMV and tumors, probably through mucosal surveillance of cellular stress, and represent a population that is largely functionally distinct from Vgamma9Vdelta2(+) T cells.     

Premature expression of T cell receptor (TCR)alphabeta suppresses TCRgammadelta gene rearrangement but permits development of gammadelta lineage T cells

The T cell receptor (TCR)gammadelta and the pre-TCR promote survival and maturation of early thymocyte precursors. Whether these receptors also influence gammadelta versus alphabeta lineage determination is less clear. We show here that TCRgammadelta gene rearrangements are suppressed in TCRalphabeta transgenic mice when the TCRalphabeta is expressed early in T cell development. This situation offers the opportunity to examine the outcome of gammadelta versus alphabeta T lineage commitment when only the TCRalphabeta is expressed. We find that precursor thymocytes expressing TCRalphabeta not only mature in the alphabeta pathway as expected, but also as CD4(-)CD8(-) T cells with properties of gammadelta lineage cells. In TCRalphabeta transgenic mice, in which the transgenic receptor is expressed relatively late, TCRgammadelta rearrangements occur normally such that TCRalphabeta(+)CD4(-)CD8(-) cells co-express TCRgammadelta. The results support the notion that TCRalphabeta can substitute for TCRgammadelta to permit a gammadelta lineage choice and maturation in the gammadelta lineage. The findings could fit a model in which lineage commitment is determined before or independent of TCR gene rearrangement. However, these results could be compatible with a model in which distinct signals bias lineage choice and these signaling differences are not absolute or intrinsic to the specific TCR structure.     

Antigen-independent appearance of recombination activating gene (RAG)-positive bone marrow B cells in the spleens of immunized mice

Splenic B lineage cells expressing recombination activation genes (RAG(+)) in mice immunized with 4-hydroxy-3-nitrophenyl-acetyl coupled to chicken gamma-globulin (NP-CGG) and the adjuvant aluminum-hydroxide (alum) have been proposed to be mature B cells that reexpress RAG after an antigen encounter in the germinal center (GC), a notion supported by findings of RAG expression in peripheral B lymphocyte populations activated in vitro. However, recent studies indicate that these cells might be immature B cells that have not yet extinguished RAG expression. Here, we employ RAG2-green fluorescent protein (GFP) fusion gene knock-in mice to show that RAG(+) B lineage cells do appear in the spleen after the administration of alum alone, and that their appearance is independent of T cell interactions via the CD40 pathway. Moreover, splenic RAG(+) B lineage cells were detectable in immunized RAG2-deficient mice adoptively transferred with bone marrow (BM) cells, but not with spleen cells from RAG(+) mice. Although splenic RAG(+) B cells express surface markers associated with GC B cells, we also find the same basic markers on progenitor/precursor BM B cells. Finally, we did not detect RAG gene expression after the in vitro stimulation of splenic RAG(-) mature B cells with mitogens (lipopolysaccharide and anti-CD40) and cytokines (interleukin [IL]-4 and IL-7). Together, our studies indicate that RAG(+) B lineage cells from BM accumulate in the spleen after immunization, and that this accumulation is not the result of an antigen-specific response.     

Activation-induced modification in the CD3 complex of the gammadelta T cell receptor

The T cell antigen receptor complexes expressed on alphabeta and gammadelta T cells differ not only in their respective clonotypic heterodimers but also in the subunit composition of their CD3 complexes. The gammadelta T cell receptors (TCRs) expressed on ex vivo gammadelta T cells lack CD3delta, whereas alphabeta TCRs contain CD3delta. While this result correlates with the phenotype of CD3delta(-/-) mice, in which gammadelta T cell development is unaffected, it is inconsistent with the results of previous studies reporting that CD3delta is a component of the gammadelta TCR. Since earlier studies examined the subunit composition of gammadelta TCRs expressed on activated and expanded peripheral gammadelta T cells or gammadelta TCR(+) intestinal intraepithelial lymphocytes, we hypothesized that activation and expansion may lead to changes in the CD3 subunit composition of the gammadelta TCR. Here, we report that activation and expansion do in fact result in the inclusion of a protein, comparable in mass and mobility to CD3delta, in the gammadelta TCR. Further analyses revealed that this protein is not CD3delta, but instead is a differentially glycosylated form of CD3gamma. These results provide further evidence for a major difference in the subunit composition of alphabeta- and gammadelta TCR complexes and raise the possibility that modification of CD3gamma may have important functional consequences in activated gammadelta T cells.     

Downmodulation of the inflammatory response to bacterial infection by gammadelta T cells cytotoxic for activated macrophages

Although gammadelta T cells are involved in the regulation of inflammation after infection, their precise function is not known. Intraperitoneal infection of T cell receptor (TCR)-delta(-/-) mice with the intracellular bacterium Listeria monocytogenes resulted in the development of necrotic foci in the livers. In contrast, the peritoneal cavities of infected TCR-delta(-/-) mice contained an accumulation of low density activated macrophages and a reduced percentage of macrophages undergoing apoptosis. gammadelta T cell hybridomas derived from mice infected with Listeria were preferentially stimulated by low density macrophages from peritoneal exudates of infected mice. Furthermore, primary splenic gammadelta T cells isolated from Listeria-infected mice were cytotoxic for low density macrophages in vitro, and cytotoxicity was inhibited in the presence of antibodies to the gammadelta TCR. These results demonstrate a novel interaction between gammadelta T cells and activated macrophages in which gammadelta T cells are stimulated by terminally differentiated macrophages to acquire cytotoxic activity and which, in turn, induce macrophage cell death. This interaction suggests that gammadelta T cells regulate the inflammatory response to infection with intracellular pathogens by eliminating activated macrophages at the termination of the response.     

Human intestinal Vdelta1+ lymphocytes recognize tumor cells of epithelial origin

gammadelta T cells can be grouped into discrete subsets based upon their expression of T cell receptor (TCR) variable (V) region families, their tissue distribution, and their specificity. Vdelta2+ T cells constitute the majority of gammadelta T cells in peripheral blood whereas Vdelta1+T cells reside preferentially in skin epithelium and in the intestine. gammadelta T cells are envisioned as first line host defense mechanisms capable of providing a source of immune effector T cells and immunomodulating cytokines such as interleukin (IL) 4 or interferon (IFN) gamma. We describe here the fine specificity of three distinct gammadelta+ tumor-infiltrating lymphocytes (TIL) obtained from patients with primary or metastatic colorectal cancer, that could be readily expanded in vitro in the presence of IL-1beta and IL-7. Irrespective of donor, these individual gammadelta T cells exhibited a similar pattern of reactivity defined by recognition of autologous and allogeneic colorectal cancer cells, renal cell cancer, pancreatic cancer, and a freshly isolated explant from human intestine as measured by cytolytic T cell responses and by IFN-gamma release. In contrast, tumors of alternate histologies were not lysed, including lung cancer, squamous cell cancer, as well as the natural/lymphocyte-activated killer cell-sensitive hematopoietic cell lines T2, C1R, or Daudi. The cell line K562 was only poorly lysed when compared with colorectal cancer targets. Target cell reactivity mediated by Vdelta1+ T cells was partially blocked with Abs directed against the TCR, the beta2 or beta7 integrin chains, or fibronectin receptor. Marker analysis using flow cytometry revealed that all three gammadelta T cell lines exhibit a similar phenotype. Analysis of the gammadelta TCR junctional suggested exclusive usage of the Vdelta1/Ddelta3/Jdelta1 TCR segments with extensive (< or = 29 bp) N/P region diversity. T cell recognition of target cells did not appear to be a major histocompatibility complex restricted or to be correlated with target cell expression of heat- shock proteins. Based on the ability of some epithelial tumors, including colorectal, pancreatic, and renal cell cancers to effectively cold target inhibit the lysis of colorectal cancer cell lines by these Vdelta1+ T cell lines, we suggest that intestinal Vdelta1+ T cell lines, we suggest that intestinal Vdelta1+ T cells are capable of recognizing cell surface Ag(s) shared by tumors of epithelial origin.     

Sublethal gamma-radiation induces differentiation of CD4-/CD8- into CD4+/CD8+ thymocytes without T cell receptor beta rearrangement in recombinase activation gene 2-/- mice

DNA recombination of the immunoglobulin (Ig) or T cell receptor (TCR) gene loci is an essential step in the production of lymphocytes bearing antigen-specific receptors. Mice that lack the ability to rearrange their Ig and TCR gene loci are devoid of mature B and T cells. Complete rearrangement and expression of the TCR-beta chain has been suggested to allow immature thymocytes to switch from the CD4-/CD8- to the CD4+/CD8+ stage of thymic development. Thus, thymocytes from severe combined immune deficient (SCID) mice or mice deficient in recombinase activation genes (RAG), which do not undergo proper DNA rearrangement, are arrested at the early CD4-/CD8- stage of development. B cell precursors in SCID or RAG mice do not progress from the B220+/sIgM- /heat stable antigen (HSA)+/CD43+ to the B220+/sIgM-/HSA+/CD43- stage. In an attempt to reconstitute RAG-2-/- mice with bone marrow- or fetal liver-derived progenitor cells, we subjected these mice to sublethal doses of gamma-radiation. It is surprising that in the absence of donor cells, irradiated RAG-2-/- mice revealed a dramatic change in their lymphoid phenotype. 14 d after irradiation, the majority of thymocytes had advanced to the CD4+/CD8+ stage of T cell development and a small number of bone marrow precursors had progressed to the CD43-, HSAhi stage of B cell development. Analysis of the resulting CD4+/CD8+ thymocytes revealed no surface expression of the TCR/CD3 complex and no V-D-J rearrangement of the TCR-beta gene locus. Our findings provide evidence for a novel pathway that allows the transition of thymocytes from the CD4-/CD8- to the CD4+/CD8+ stage and that does not appear to require TCR-beta chain rearrangement.     

Generation of artificial lymph nodes (aLNs) and their immunological function

We demonstrated that artificial lymph nodes (aLNs) could be generated by the transplantation of stromal cell-embedded biocompatible scaffolds into the renal subcapsular space in mice. T and B cell domains that form in the aLNs have a similar function in the immune response as the follicles of normal lymphoid tissue. The aLNs were transplantable to naive normal as well as to severe combined immunodeficient (SCID) or RAG deficient mice where aLNs efficiently induced secondary immune responses. Antigen-specific secondary responses were strongly induced in aLNs even four weeks after their transplantation. The antigen-specific antibody responses in the lymphocyte-deficient SCID or RAG KO mice carrying aLNs were enormous, reaching 7-8mg/mL in serum. The cells from the aLNs migrated to the SCID or RAG KO mouse spleen and bone marrow where they expanded to generate large numbers of antigen-specific IgG-class antibody-forming cells. Secondary responses were maintained with time after antigen challenge, indicating that aLNs can support memory B cells and long lived plasma cells. To our surprise, memory type CD4+ T cells (CD44(hi), CD62L(lo)) were highly enriched in the aLNs as well as in spleens of SCID mice carrying the aLNs. These data indicate that the lymph node-like lymphoid tissues which were generated on stromal cell-embedded biocompatible scaffolds were easily transplantable and could mount a strong antigen specific humoral as well as cellular immune responses.     

Flow cytometric assessment of autologous gammadelta T cells in patients with acute myeloid leukemia: potential effector cells for immunotherapy?

BACKGROUND: Gammadelta T cells are a rare component of the circulating innate immune system capable of exerting anti-neoplastic activity. This population may be suitable for the adoptive immunotherapy of acute myeloid leukemia (AML). Little is known however, about the frequency and function of circulating gammadelta T cells in AML. The aim of the study was to enumerate peripheral blood gammadelta T cells in patients with AML and explore the feasibility of their use clinically. METHODS: We compared the absolute circulating gammadelta T cell levels in 33 AML patients before and after treatment versus 20 healthy volunteers using flow cytometry. The function of gammadelta T cells was assessed by detection of intracelluar interferon-gamma (IFN-gamma) and cytotoxicity against leukemic blasts. RESULTS: AML patients with high blast counts prior to induction chemotherapy had marginally decreased gammadelta T cell levels compared with healthy controls: median 38/microL versus 83/microL; P = 0.051. Sequential gammadelta T cell enumeration after induction showed significantly decreased counts in patients with a persistently high blast burden compared to patients with reduced but detectable residual disease (molecular maker or borderline bone marrow infiltration): median 7/microL versus 105/microL; P = 0.008. Patients with residual disease had significantly higher gammadelta T cell counts compared to those retested after they had achieved complete remission (CR); P = 0.0025. In CR, gammadelta T cell counts remained lower than those of healthy individuals: median 33/microL versus 83/microL, P = 0.030. We detected a sharp increase (on average, four-fold higher than values in CR) of gammadelta T cells in patients in very early morphologic or molecular relapse. We also tested the functional properties of gammadelta T cells from patients with AML in CR. Flow cytometric assessment of IFN-gamma revealed similar numbers of gammadelta T cells expressing the T1 cytokine compared with healthy controls. We also showed that gammadelta T cells were able to kill leukemic target cells in vitro. CONCLUSION: Flow cytometric assessment of gammadelta T cells in patients with AML revealed quantitative shifts with respect to disease status. Our data suggest that gammadelta T cells warrant further investigation as potential therapeutic agents.     

Disruption of the Bcl6 Gene Results in an Impaired Germinal Center Formation

The Bcl6 gene has been identified from the chromosomal translocation breakpoint in B cell lymphomas, and its products are expressed highly in germinal center (GC) B cells. To investigate the function of Bcl6 in lymphocytes, we have generated RAG1-deficient mice reconstituted with bone marrow cells from Bcl6-deficient mice (Bcl6^−/−RM). Lymphogenesis in primary lymphoid tissues of Bcl6^−/−RM is normal, and Bcl6^−/−RM produced control levels of primary IgG1 antibodies specific to T cell–dependent antigens. However, GCs were not found in these mice. This defect was mainly due to the abnormalities of B cells. Therefore, Bcl6 is essential for the differentiation of GC B cells.     

CD4 expression is differentially required for deletion of MLS-1a- reactive T cells

Clonal deletion of thymocytes expressing potentially self-reactive T cell receptors (TCRs) occurs during thymocyte ontogeny. Mice deficient for CD4 expression provide a unique model system to study the contribution of the CD4 molecule in negative selection of T cells reactive against the major histocompatibility complex class II- associated retroviral self-superantigen, Mls-1a. In the presence of Mls- 1a determinants, mature CD8+ T cells expressing V beta 6, 8.1, and 9 were deleted in CD4-deficient mice, thus demonstrating that TCR affinity for Mls-1a is sufficient for deletion and that a signal through CD4 was not required. However, in instances where the TCR affinity for Mls-1a is low, as in the case of V beta 7+ T cells, CD4 expression was required for clonal deletion. These results demonstrate that for Mls-1a-mediated clonal deletion of T cells, the requirement for the accessory or coreceptor function of CD4 depends on the affinity of the TCR.     

T cell-independent antibody-mediated clearance of polyoma virus in T cell-deficient mice

Polyomavirus (PyV) infection of SCID mice, which lack functional T and B cells, leads to a lethal acute myeloproliferative disease (AMD) and to high levels of virus replication in several organs by two wk after infection. This is in contrast to infection of T cell-deficient athymic nude mice, which are resistant to acute PyV-induced disease and poorly replicate the virus in their organs. This major difference in the virus load and in the outcome of PyV infection between SCID and nude mice suggested that an efficient, T cell-independent antiviral mechanism operates in T cell-deficient, PyV infected mice. To investigate this possibility, mice with different genetically engineered T and/or B cell deficiencies and SCID mice adoptively reconstituted with B and/or T cells were infected with PyV. The results indicated that the presence of B cells in the absence of T cells protected mice from the AMD, and this was accompanied by a major reduction of PyV in all organs tested. Sera from PyV-infected T cell receptor (TCR) alpha beta knockout or TCR alpha beta gamma delta knockout mice contained IgG2a antibodies to PyV. Sera or purified immunoglobulin fractions from PyV-infected TCR alpha beta knockout mice protected SCID mice from the PyV-induced AMD. To our knowledge, this is the first report of an effective T cell-independent antibody response clearing a virus and changing the outcome of infection from 100% mortality to 100% survival.     

Immunization with soluble BDC 2.5 T cell receptor-immunoglobulin chimeric protein:antibody specificity and protection of nonobese diabetic mice against adoptive transfer of diabetes by maternal immunization

The BDC 2.5 T cell clone is specific for pancreatic beta-cell antigen presented by I-Ag7, and greatly accelerates diabetes when injected into 10-21-d-old nonobese diabetic (NOD) mice. The BDC 2.5 T cell receptor (TCR) has been solubilized as a TCR-IgG1 chimeric protein. All NOD mice immunized against BDC 2.5 TCR-IgG1 produced antibodies recognizing TCR C alpha/C beta epitopes that were inaccessible on the T cell surface. 56% of the mice produced antibodies against the BDC 2.5 clonotype that specifically blocked antigen activation of BDC 2.5 cells. We have used the adoptive transfer model of diabetes to demonstrate that maternal immunization with soluble TCR protects young mice from diabetes induced by the BDC 2.5 T cell clone.