T-cell Antigen Receptor Expression Research Articles

Trogocytic molting of T cell microvilli upregulates T cell receptor surface expression and promotes clonal expansion

Although T cell activation is known to involve the internalization of the T cell antigen receptor (TCR), much less is known regarding the release of TCRs following T cell interaction with cognate antigen-presenting cells. In this study, we examine the physiological mechanisms underlying TCR release following T cell activation. We show that T cell activation results in the shedding of TCRs in T cell microvilli, which involves a combined process of trogocytosis and enzymatic vesiculation, leading to the loss of membrane TCRs and microvilli-associated proteins and lipids. Surprisingly, unlike TCR internalization, this event results in the rapid upregulation of surface TCR expression and metabolic reprogramming of cholesterol and fatty acid synthesis to support cell division and survival. These results demonstrate that TCRs are lost through trogocytic ‘molting’ following T cell activation and highlight this mechanism as an important regulator of clonal expansion.

Genetic tracing of thymic T cell differentiation

Abstract Thymic T cell differentiation is controlled by stereotyped processes that remain obscure. Here we develop a novel mouse genetic tool to trace thymic T cells during T cell antigen receptor (TCR) rearrangement. Briefly, we insert inducible CreER into the Rag1 locus that is transiently expressed at early thymic T cell differentiation. In the presence of Rosa reporters, Rag1-CreER mice enable us to irreversibly label T cells between the double negative (DN) and double positive (DP) stages upon acute tamoxifen treatment. In the physiological setting and absence of the perturbations historically used to investigate thymic T cell differentiation, our genetic tracing in combination with flow cytometric measurement of cell fates and functional states maps the kinetics, stoichiometry, and cell fate determination of thymic T cells. We further integrate genetic tracing experiments with unbiased single cell RNA sequencing, TCR sequencing, and the expression of cell surface markers, linking thymic T cell differentiation stages to dynamic gene expression. In particular, we show that regulatory T cells acquire CD73 expression over time as a potential consequence of persistent TCR signaling and expression of lineage determinant Foxp3, marking a maturation process during lineage commitment and functional adaptation. Taken together, our integrated genetic tracing of thymic T cells resolves complex differentiation processes coupled with the alterations of gene expression, selection, and cell fate determination. Our results also serve as a foundation for further exploration of the thymic T cell states, developmental pathways, drivers, and modulators involved in these crucial biological processes. R21 AI146614

Single-cell transcriptome landscape and antigen receptor dynamic during SARS-CoV-2 vaccination

Vaccination by inactivated vaccine is an effective strategy to prevent the COVID-19 pandemic. However, the detailed molecular immune response at single-cell level is poorly understood. In this study, we systematically delineated the landscape of the pre- and post-vaccination single-cell transcriptome, TCR (T cell antigen receptor) and BCR (B cell antigen receptor) expression profile of vaccinated candidates. The bulk TCR sequencing analysis of COVID-19 patients was also performed. Enrichment of a clonal CD8+ T cell cluster expressing specific TCR was identified in both vaccination candidates and COVID-19 patients. These clonal CD8+ T cells showed high expression of cytotoxicity, phagosome and antigen presentation related genes. The cell–cell interaction analysis revealed that monocytes and dendritic cells could interact with these cells and initiate phagocytosis via ICAM1-ITGAM and ITGB2 signaling. Together, our study systematically deciphered the detailed immunological response during SARS-CoV-2 vaccination and infection. It may facilitate understanding the immune response and the T-cell therapy against COVID-19.

The role of the different CD3γ domains in TCR expression and signaling.

The CD3 subunits of the T-cell antigen receptor (TCR) play a central role in regulation of surface TCR expression levels. Humans who lack CD3γ (γ—) show reduced surface TCR expression levels and abolished phorbol ester (PMA)-induced TCR down-regulation. The response to PMA is mediated by a double leucine motif in the intracellular (IC) domain of CD3γ. However, the molecular cause of the reduced TCR surface expression in γ— lymphocytes is still not known. We used retroviral vectors carrying wild type CD3γ or CD3δ or the following chimeras (EC-extracellular, TM-transmembrane and IC): δECγTMγIC (δγγ for short), γγδ, γδδ and γγ-. Expression of γγγ, γγδ, γδδ or γγ- in the γ— T cell line JGN, which lacks surface TCR, demonstrated that cell surface TCR levels in JGN were dependent on the EC domain of CD3γ and could not be replaced by the one of CD3δ. In JGN and primary γ— patient T cells, the tested chimeras confirmed that the response to PMA maps to the IC domain of CD3γ. Since protein homology explains these results better than domain structure, we conclude that CD3γ contributes conformational cues that improve surface TCR expression, likely at the assembly or membrane transport steps. In JGN cells all chimeric TCRs were signalling competent. However, an IC domain at CD3γ was required for TCR-induced IL-2 and TNF-α production and CD69 expression, indicating that a TCR without a CD3γ IC domain has altered signalling capabilities.

Early expression of mature αβ TCR in CD4−CD8− T cell progenitors enables MHC to drive development of T-ALL bearing NOTCH mutations

During normal T cell development in mouse and human, a low-frequency population of immature CD4-CD8- double-negative (DN) thymocytes expresses early, mature αβ T cell antigen receptor (TCR). We report that these early αβ TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αβ T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αβ TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors invivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.

Public and private human T-cell clones respond differentially to HCMV antigen when boosted by CD3 copotentiation

AbstractHuman cytomegalovirus (HCMV) induces long-lasting T-cell immune responses that control but do not clear infection. Typical responses involve private T-cell clones, expressing T-cell antigen receptors (TCRs) unique to a person, and public T-cell clones with identical TCRs active in different people. Here, we report the development of a pretherapeutic immunostimulation modality against HCMV for human T cells, CD3 copotentiation, and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 copotentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was coengaged with antigen. When applied in recall assays, CD3 copotentiation enhanced the expansion of both public and private T-cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLVPMVATV (NLV) peptide from HCMV pp65 protein. Interestingly, public vs private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmacoimmunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 copotentiation of T-cell clones from HLA-A2(−) donors and 1 HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T-cell copotentiation can increase the expansion of different classes of T-cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV.

Aberrantly Expressed Embryonic Protein NODAL Alters Breast Cancer Cell Susceptibility to γδ T Cell Cytotoxicity.

Gamma delta (γδ) T cells kill transformed cells, and increased circulating γδ T cells levels correlate with improved outcome in cancer patients; however, their function within the breast tumor microenvironment (TME) remains controversial. As tumors progress, they begin to express stem-cell associated proteins, concomitant with the emergence of therapy resistant metastatic disease. For example, invasive breast cancers often secrete the embryonic morphogen, NODAL. NODAL has been shown to promote angiogenesis, therapy resistance and metastasis in breast cancers. However, to date, little is known about how this secreted protein may interact with cells in the TME. Herein we explore how NODAL in the TME may influence γδ T cell function. We have assessed the proximity of γδ T cells to NODAL in a cohort of triple negative breast tumors. In all cases in which γδ T cells could be identified in these tumors, γδ T cells were found in close proximity to NODAL-expressing tumor cells. Migration of γδ and αβ T cells was similar toward MDA-MB-231 cells in which NODAL had been knocked down (shN) and MDA-MB-231 scrambled control cells (shC). Furthermore, Vδ1 γδ T cells did not migrate preferentially toward conditioned medium from these cell lines. While 24-h exposure to NODAL did not impact CD69, PD-1, or T cell antigen receptor (TCR) expression on γδ T cells, long term exposure resulted in decreased Vδ2 TCR expression. Maturation of γδ T cells was not significantly influenced by NODAL stimulation. While neither short- nor long-term NODAL stimulation impacted the ability of γδ T cells to kill MCF-7 breast cancer cells, the absence of NODAL resulted in greater sensitivity of targets to γδ T cell cytotoxicity, while overexpression of NODAL conferred resistance. This appeared to be at least in part due to an inverse correlation between NODAL and surface MICA/B expression on breast cancer target lines. As such, it appears that NODAL may play a role in strategies employed by breast cancer cells to evade γδ T cell targeting, and this should be considered in the development of safe and effective γδ T cell immunotherapies.

A New Mechanism for T-ALL Leukemogenesis: Early Expression of Alpha-Beta TCR Occurs in a Natural Subset of CD4-CD8- Double Negative (DN) Thymocytes Where MHC Engagement May Drive NOTCH Mutation and Tumor Initiation

T cell lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from transformed thymocytes. Most human T-ALL involves hyperactive NOTCH signaling that is often caused by activating NOTCH mutations. However, the identification of specific molecular signals that might induce or select for mutation and transformation are incompletely understood. We report that an understudied low-frequency, natural thymocyte subset expresses αβ T cell antigen receptor (TCR) earlier than most cells in mice and humans; engagement of the early αβTCR by major histocompatibility complexes (MHC) can cause outgrowth of NOTCH1 mutant clones and T-ALL leukemogenesis in a mouse model of T-ALL. Assessment of 5 recent human T-ALL cases found one to present this unique CD4-CD8- double- negative (DN) stage as the earliest identifiable developmental stage. These studies present a model of T-ALL leukemogenesis that identifies (i) a natural cell stage of origin susceptible to transformation, (ii) a matching mouse model showing that a signaling receptor (αβTCR) and its ligand (MHC) drive leukemogeneis and outgrowth of tumors bearing activating NOTCH1 mutations, and (iii) a human case that presents with a tumor consistent with this model and mechanism. In past work, the pre-TCR has been shown to impact T-ALL development in mice (Campese et al, Blood 2006), but an oncogenic role for the mature αβTCR is less well characterized and somewhat surprising. This is because, although T-ALL tumor cells may express variable levels of surface αβTCR/CD3, the earliest cell stages that are thought to transform are also thought to precede stages with αβTCR expression. Most conventional αβ thymocytes rearrange TCRβ and TCRα loci in separate, ordered developmental stages. However, some thymocytes in the conventional pathway rearrange both at DN stage thus exhibiting 'precocious' αβTCR (PAT) expression. Importantly, these PAT cells are indeed part of the conventional αβ lineage, being a 'subset' only due to early αβTCR expression but without known distinction in ultimate immune function (Aifantis et al, JEM 2006). We found that ~0.01% of mouse and human thymocytes are such PAT cells at steady state. To interrogate the PAT thymocyte surface phenotype, we performed multi-parametric flow cytometry with Spanning-tree Progression Analysis of Density-normalized Events (SPADE). This revealed that PAT thymocytes constitute a DN subset that is not associated with other well-described unconventional DN thymocytes known to express αβTCR, consistent with as the expectation that PATs are part of the conventional developmental pathway. We observed that the OT1 TCR transgene is expressed in mice with parallel timing and level to the natural PAT subset, allowing use of this model to study antigen-dependent signaling and oncogenesis. In a cohort study, no T-ALL was observed in wild-type C57BL/6 or OT-1.β2M-/- mice (deficient in endogenous antigen presentation), but MHC-sufficient OT-1 mice developed PAT-stage-specific T-ALL with activating NOTCH1 mutations. Transplant experiments corroborated a requirement for antigen presentation and TCR signaling for tumor maintenance as transplanted tumors grew in MHC+ but not MHC-deficient mice. This predicted that PAT thymocytes might have an unusual ability to signal through αβTCR even without coreceptor expression. When cultured in the presence of either exogenously added β2M or antigen presenting cells, both untransformed and neoplastic PAT cells upregulated CD69 in response to the OT-1 antigenic peptide, OVA. Furthermore, ex vivo analysis of PAT cells from polyclonal C57BL/6 versus MHC-deficient mice showed intrinsic upregulation of TCR-signaling-dependent Nur77 in an MHC-dependent manner. These data revealed a unique ability of PAT cells to engage in co-receptor independent but antigen-dependent signaling. Microarray analysis showed that the gene expression profile of neoplastic PAT cells from OT-1 T-ALL most closely resembled that of conventional post β-selection DN thymocytes, in agreement with the natural PAT stage during normal T cell development. These data support a model in which transformation occurred in the naturally occurring αβ PAT thymocyte subset as cell-of-origin. Collectively, our data suggest that precocious αβTCR expression and coreceptor-independent antigen engagement can cause activating NOTCH mutation and T-ALL development. Disclosures No relevant conflicts of interest to declare.

Analysis of the recovery of CD247 expression in a PID patient: insights into the spontaneous repair of defective genes

AbstractMutations in T-cell antigen receptor (TCR) subunit genes cause rare immunodeficiency diseases characterized by impaired expression of the TCR at the cell surface and selective T lymphopenia. Here, detailed analyses of spontaneously arising somatic mutations that recover CD247, and thus TCR expression, in a newly identified CD247-deficient patient are described. The recovery of CD247 expression in some patient T cells was associated with both reversion of the inactivating mutation and a variant with a compensating mutation that could reconstitute TCR expression, but not as efficiently as wild-type CD247. Multiple mutations were found in CD247 complementary DNAs (cDNAs) cloned from the patient as well as in cDNA and genomic DNA from other individuals, suggesting that genetic variation in this gene is frequent. Analyses of other genes mutated in primary immunodeficiency diseases (PIDs) where reversions have been described also revealed a higher rate of mutation than that observed for genes mutated in PIDs where revertants have not been identified or control genes. These data support the hypothesis that the occurrence of somatic mutations that may reconstitute genetic defects in PID is related to an increased propensity of those genes to mutate.

TCR signal strength controls thymic differentiation of discrete proinflammatory γδ T cell subsets.

The murine thymus produces discrete γδ T cell subsets making either interferon-γ (IFN--γ) or interleukin 17 (IL-17), but the role of the TCR in this developmental process remains controversial. Here we show that mice haploinsufficient for both Cd3g and Cd3d (CD3DH, for CD3 double haploinsufficient) have reduced TCR expression and signaling strength selectively on γδ T cells. CD3DH mice had normal numbers and phenotype of αβ thymocyte subsets but impaired differentiation of fetal Vγ6+ (but not Vγ4+) IL-17-producing γδ T cells and a marked depletion of IFN-γ-producing CD122+ NK1.1+ γδ T cells throughout ontogeny. Adult CD3DH mice showed reduced peripheral IFN-γ+ γδ T cells and were resistant to experimental cerebral malaria. Thus, TCR signal strength within specific thymic developmental windows is a major determinant of the generation of proinflammatory γδ T cell subsets and their impact on pathophysiology.

Control of T cell antigen reactivity via programmed TCR downregulation.

The T cell receptor (TCR) is unique in that its affinity for ligand is unknown prior to encounter and can vary by orders of magnitude. How the immune system regulates individual T cells that display highly different reactivity to antigen remains unclear. Here we identified that activated CD4+ T cells, at the peak of clonal expansion, persistently downregulate TCR expression in proportion to the strength of initial antigen recognition. This programmed response increases the threshold for cytokine production and recall proliferation in a clone-specific manner, ultimately excluding clones with the highest antigen reactivities. Thus, programmed TCR downregulation represents a negative feedback mechanism to constrain T cell effector function with a suitable time delay, thereby allowing pathogen control while avoiding excess inflammatory damage.

Butyrophilin 3A1 binds phosphorylated antigens and stimulates human γδ T cells

Human T cells that express a T cell antigen receptor (TCR) containing γ-chain variable region 9 and δ-chain variable region 2 (Vγ9Vδ2) recognize phosphorylated prenyl metabolites as antigens in the presence of antigen-presenting cells but independently of major histocompatibility complex (MHC), the MHC class I-related molecule MR1 and antigen-presenting CD1 molecules. Here we used genetic approaches to identify the molecule that binds and presents phosphorylated antigens. We found that the butyrophilin BTN3A1 bound phosphorylated antigens with low affinity, at a stoichiometry of 1:1, and stimulated mouse T cells with transgenic expression of a human Vγ9Vδ2 TCR. The structures of the BTN3A1 distal domain in complex with host- or microbe-derived phosphorylated antigens had an immunoglobulin-like fold in which the antigens bound in a shallow pocket. Soluble Vγ9Vδ2 TCR interacted specifically with BTN3A1-antigen complexes. Accordingly, BTN3A1 represents an antigen-presenting molecule required for the activation of Vγ9Vδ2 T cells.

Human CD3γ, but not CD3δ, haploinsufficiency differentially impairs γδ versus αβ surface TCR expression

BackgroundThe T cell antigen receptors (TCR) of αβ and γδ T lymphocytes are believed to assemble in a similar fashion in humans. Firstly, αβ or γδ TCR chains incorporate a CD3δε dimer, then a CD3γε dimer and finally a ζζ homodimer, resulting in TCR complexes with the same CD3 dimer stoichiometry. Partial reduction in the expression of the highly homologous CD3γ and CD3δ proteins would thus be expected to have a similar impact in the assembly and surface expression of both TCR isotypes. To test this hypothesis, we compared the surface TCR expression of primary αβ and γδ T cells from healthy donors carrying a single null or leaky mutation in CD3G (γ+/−) or CD3D (δ+/−, δ+/leaky) with that of normal controls.ResultsAlthough the partial reduction in the intracellular availability of CD3γ or CD3δ proteins was comparable as a consequence of the mutations, surface TCR expression measured with anti-CD3ε antibodies was significantly more decreased in γδ than in αβ T lymphocytes in CD3γ+/− individuals, whereas CD3δ+/− and CD3δ+/leaky donors showed a similar decrease of surface TCR in both T cell lineages. Therefore, surface γδ TCR expression was more dependent on available CD3γ than surface αβ TCR expression.ConclusionsThe results support the existence of differential structural constraints in the two human TCR isotypes regarding the incorporation of CD3γε and CD3δε dimers, as revealed by their discordant surface expression behaviour when confronted with reduced amounts of CD3γ, but not of the homologous CD3δ chain. A modified version of the prevailing TCR assembly model is proposed to accommodate these new data.

Human Vδ1 γδ T cells expanded from peripheral blood exhibit specific cytotoxicity against B-cell chronic lymphocytic leukemia-derived cells

There is increasing interest in using γδ T cells (GDTC) for cancer immunotherapy. Most studies have been concerned with the Vδ2 subset in blood, for which several expansion protocols exist. We have developed a protocol to expand Vδ1 and Vδ2 preferentially from human blood. We have characterized these subsets and their specificities for leukemic targets. GDTC were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors via positive magnetic cell sorting; their proliferation in vitro was induced by exposure to the mitogen concanavalin A (Con A). CD107 and cytotoxicity (Cr(51)-release and flow cytometric) assays were performed. GDTC clones and target cells were immunophenotyped via flow cytometry. Longer initial exposure to Con A typically resulted in higher Vδ1 prevalence. Vδ1 were activated by and cytotoxic to B-cell chronic lymphocytic leukemia (B-CLL)-derived MEC1 cells, whereas Vδ2 also responded to MEC1 but more so to the Philadelphia chromosome-positive [Ph+] leukemia cell line EM-enhanced green fluorescent protein (2eGFPluc). Vδ2 clone cytotoxicity against EM-2eGFPluc correlated with Vδ2 T-cell antigen receptor (TCR) and receptor found on Natural Killer cells and many T-cells (NKG2D), whereas Vδ1 clone cytotoxicity versus MEC1 correlated with Vδ1 TCR, CD56 and CD95 expression. Vδ1 also killed Epstein-Barr Virus (EBV)-negative B-CLL-derived TMD2 cells. Immunophenotyping revealed reduced HLA-ABC expression on EM-2eGFPluc, whereas MEC1 and TMD2 exhibited higher Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAILR1). Our ability to expand peripheral Vδ1 cells and show their cytotoxicity to B-CLL-derived cell lines suggests that this novel approach to the cellular treatment of B-CLL may be feasible.

A Lysosomal Protein Negatively Regulates Surface T Cell Antigen Receptor Expression by Promoting CD3ζ-Chain Degradation

Modulation of surface T cell antigen receptor (TCR) expression is an important mechanism for the regulation of immune responses and the prevention of T cell hyperactivation and autoimmunity. The TCR is rapidly internalized after antigen stimulation and then degraded in lysosomes. However, few of the molecules involved in this process have been identified. We demonstrate that the lysosomal protein LAPTM5 negatively regulated surface TCR expression by specifically interacting with the invariant signal-transducing CD3zeta chain and promoting its degradation without affecting other CD3 proteins, CD3epsilon, CD3delta, or CD3gamma. TCR downmodulation required the polyproline-tyrosine motifs and the ubiquitin-interacting motif of LAPTM5. LAPTM5 deficiency resulted in elevated TCR expression on both CD4(+)CD8(+) thymocytes and spleen T cells after CD3 stimulation, as well as enhanced T cell responses in vitro and in vivo. These results identify a lysosomal protein important for CD3zeta degradation and illustrate a unique mechanism for the control of surface TCR expression and T cell activation.

The proline-rich sequence of CD3ε controls T cell antigen receptor expression on and signaling potency in preselection CD4+CD8+ thymocytes

Antigen recognition by T cell antigen receptors (TCRs) is thought to 'unmask' a proline-rich sequence (PRS) present in the CD3epsilon cytosolic segment, which allows it to trigger T cell activation. Using 'knock-in' mice with deletion of the PRS, we demonstrate here that elimination of the CD3epsilon PRS had no effect on mature T cell responsiveness. In contrast, in preselection CD4+CD8+ thymocytes, the CD3epsilon PRS acted together with the adaptor protein SLAP to promote CD3zeta degradation, thereby contributing to downregulation of TCR expression on the cell surface. In addition, analysis of CD4+CD8+ thymocytes of TCR-transgenic mice showed that the CD3epsilon PRS enhanced TCR sensitivity to weak ligands. Our results identify previously unknown functions for the evolutionarily conserved CD3epsilon PRS at the CD4+CD8+ developmental stage and suggest a rather limited function in mature T cells.

Effects of immunosuppressive treatment in prevention of calcification in aortic valved homograft: experiment with rats

To investigate the effects of immunosuppressive treatment in prevention of calcification in aortic valved homograft (AVH). 120 Wistar rats were randomly divided into 4 equal groups: Group A (allogene group) undergoing incision of the abdominal aorta and implantation of the AVH with myocardial cuff from SD rats, Group B, injected with cyclosporine A intraperitoneally one day after the implantation, Group C, injected intraperitoneally with anti-dendritic cell monoclonal antibody (DCmAb) one day after the implantation, and Group D (isogenenic or control group), receiving the AVH of another Wistar rats. All groups were further subdivided into 5 equal subgroups to be sacrificed at different time points: 2, 4, 8, 12, and 16 weeks postoperatively. Blood samples were obtained from the vena cava to detect the expression of T-cell antigen receptor (TCR)-alpha and beta and CD28 by flow cytometry. AVH specimens were obtained to observe the changes of endotheliocytes and smooth muscle cells with light and electron microscopy. The expression of CD54 was detected by immunohistochemistry. The calcium content of the AVH tissue after transplantation was examined by flame atomic absorption spectrophotometry. (1) Compared with the isogenic group, the expression levels of TCR-alpha and beta and CD28 in the allogener groups were all significantly higher at all time points (all P < 0.01), peaked 2 approximately 4 weeks after operation, then gradually decreased, and approached the level of the controls 12 weeks after the implantation. Specifically, the expression levels of TCR-alpha and TCR-beta 2 and 4 weeks postoperatively of Group A were 52.4% +/- 3.3% and 43.8% +/- 6.4% respectively, significantly higher than those of Group B [(34.5 +/- 3.5)% and (31.6 +/- 2.6)% respectively], Group C [(31.6 +/- 2.3)% and (29.5 +/- 3.0)% respectively), and Group D (23.2 +/- 1.3)% and (21.6 +/- 2.3)% (all P < 0.01)]; and the CD28 expression level 2 approximately 4 weeks after operation of Group A were (51.7 +/- 7.5)% and (66.3 +/- 4.4)% respectively, both significantly higher than those of Group B [(41.2 +/- 1.6)% and (55.1 +/- 5.1)% respectively], Group C [(36.6 +/- 3.6)% and (51.8 +/- 5.6)% respectively], and Group D [30.7 +/- 1.4)% and (33.3 +/- 0.9)% respectively)] [all P < 0.01 except those levels 12 and 16 weeks after the operation in each subgroup (P > 0.05)] And the levels of TCR-alpha and TCR-beta and CD28 of the 2 treatment groups were all significantly lower than those of the untreated group (Group A) (all P < 0.01). (2) The calcium contents of the AVH tissues of Group A, B, and C significantly increased 4 weeks after the operation and peaked 12 and 16 weeks after operation. No significant difference in calcium level was found in Group D at different time points (all P > 0.05). The calcium contents in AVH tissues 4 and 8 weeks postoperatively of Groups A, B, and C were (2856 +/- 79) microg/g and (3587 +/- 168) microg/g respectively, (2518 +/- 73) microg/g, (3237 +/- 187) microg/g; and (2176 +/- 210) microg/g and (3089 +/- 176) microg/g; all significantly higher than those of Group D (860 +/- 60) microg/g and (870 +/- 50) microg/g respectively, all P < 0.01. Immunosuppressive treatment obviously reduces the immune rejection and delays the course of AVH calcification.

T−B+NK+ severe combined immunodeficiency caused by complete deficiency of the CD3ζ subunit of the T-cell antigen receptor complex

AbstractCD3ζ is a subunit of the T-cell antigen receptor (TCR) complex required for its assembly and surface expression that also plays an important role in TCR-mediated signal transduction. We report here a patient with T−B+NK+ severe combined immunodeficiency (SCID) who was homozygous for a single C insertion following nucleotide 411 in exon 7 of the CD3ζ gene. The few T cells present contained no detectable CD3ζ protein, expressed low levels of cell surface CD3ε, and were nonfunctional. CD4+CD8−CD3εlow, CD4−CD8+CD3εlow, and CD4−CD8−CD3εlow cells were detected in the periphery, and the patient also exhibited an unusual population of CD56−CD16+ NK cells with diminished cytolytic activity. Additional studies demonstrated that retrovirally transduced patient mutant CD3ζ cDNA failed to rescue assembly of nascent complete TCR complexes or surface TCR expression in CD3ζ-deficient MA5.8 murine T-cell hybridoma cells. Nascent transduced mutant CD3ζ protein was also not detected in metabolically labeled MA5.8 cells, suggesting that it was unstable and rapidly degraded. Taken together, these findings provide the first demonstration that complete CD3ζ deficiency in humans can cause SCID by preventing normal TCR assembly and surface expression.

T-B+NK+ Severe Combined Immunodeficiency Caused by Complete Deficiency of the CD3zeta Subunit of the T Cell Antigen Receptor Complex

RATIONALE: CD3ζɛτα is a subunit of the T cell antigen receptor (TCR) complex required for its assembly and surface expression that also plays an important role in TCR-mediated signal transduction. Hence, we hypothesized complete CD3ζɛτα deficiency would result in severe combined immunodeficiency (SCID). METHODS: TCR complex expression was analyzed in purified CD3ɛπσιλoνlow cells from a patient with T-B+NK+ SCID. Patient lymphocyte subsets were analyzed by flow cytometry. CD3ζɛτα gene mutation analysis was performed by automated DNA sequencing. Additional studies included analysis of TCR expression in retrovirally-transduced CD3ζɛτα-deficient MA5.8 cells. RESULTS: The patient was homozygous for a deleterious mutation in the CD3ζɛτα gene. The few T cells present contained no detectable CD3ζɛτα protein or complete TCR complexes, and only expressed low levels of incomplete TCR complexes on the cell surface that contained CD3ɛπσιλoν in the absence of TCRαλπηαβɛτα or TCRgammadelta and were non-functional. The patient also expressed large numbers of CD3epsilonlow CD4-CD8- cells in the periphery, and exhibited an unusual population of CD56-CD16+ NK cells with diminished cytolytic activity. Retrovirally-transduced patient mutant CD3zeta cDNA failed to rescue assembly of nascent complete TCR complexes or surface TCR expression in CD3zeta-deficient MA5.8 murine T cell hybridoma cells. Nascent transduced mutant CD3zeta protein was also not detected in metabolically labeled MA5.8 cells, demonstrating that it was unstable and rapidly degraded. CONCLUSION: Taken together, these findings provide the first demonstration that complete CD3ζɛτα deficiency in humans can cause SCID by preventing assembly and surface expression of complete TCR complexes.

Infiltrating T-lymphocyte Receptor Vβ Gene Family Utilization in Autoimmune Thyroid Disease

The expression of T-cell antigen receptor (TCR) Vbeta genes in autoimmune thyroid diseases (AITDs) was investigated. RNA was extracted from the thyroid tissue of 23 patients with early-stage Graves' disease, 19 patients with late-stage Graves' disease and 20 patients with Hashimoto's disease. Peripheral blood lymphocytes from patients and 20 normal subjects (controls) were analysed in parallel. AITD was found to be associated with diminished TCR Vbeta gene family utilization. In addition, AITDs appeared to select for specific TCR Vbeta families. The Vbeta3, Vbeta5 and Vbeta8 families were expressed more frequently in thyroid-infiltrating T-lymphocytes of early-stage Graves' disease than other Vbeta gene families. Selective expression was not observed in infiltrating T-lymphocytes obtained from thyroid tissue of patients with late-stage Graves' disease or Hashimoto's disease. Preferentially expressed TCR Vbeta gene families may be useful as molecular targets for targeted immunotherapy of AITDs.