Edgar Fernández-Malavé

Different composition of the human and the mouse γδ T cell receptor explains different phenotypes of CD3γ and CD3δ immunodeficiencies

The γδ T cell receptor for antigen (TCR) comprises the clonotypic TCRγδ, the CD3 (CD3γε and/or CD3δε), and the ζζ dimers. γδ T cells do not develop in CD3γ-deficient mice, whereas human patients lacking CD3γ have abundant peripheral blood γδ T cells expressing high γδ TCR levels. In an attempt to identify the molecular basis for these discordant phenotypes, we determined the stoichiometries of mouse and human γδ TCRs using blue native polyacrylamide gel electrophoresis and anti-TCR–specific antibodies. The γδ TCR isolated in digitonin from primary and cultured human γδ T cells includes CD3δ, with a TCRγδCD3ε2δγζ2 stoichiometry. In CD3γ-deficient patients, this may allow substitution of CD3γ by the CD3δ chain and thereby support γδ T cell development. In contrast, the mouse γδ TCR does not incorporate CD3δ and has a TCRγδCD3ε2γ2ζ2 stoichiometry. CD3γ-deficient mice exhibit a block in γδ T cell development. A human, but not a mouse, CD3δ transgene rescues γδ T cell development in mice lacking both mouse CD3δ and CD3γ chains. This suggests important structural and/or functional differences between human and mouse CD3δ chains during γδ T cell development. Collectively, our results indicate that the different γδ T cell phenotypes between CD3γ-deficient humans and mice can be explained by differences in their γδ TCR composition.

TCR signal strength controls thymic differentiation of discrete proinflammatory [gamma][delta] T cell subsets

The mouse thymus produces discrete γδ T cell subsets that make either interferon-γ (IFN-γ) or interleukin 17 (IL-17), but the role of the T cell antigen receptor (TCR) in this developmental process remains controversial. Here we show that Cd3g+/− Cd3d+/− (CD3 double-haploinsufficient (CD3DH)) mice have reduced TCR expression and signaling strength on γδ T cells. CD3DH mice had normal numbers and phenotypes 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.

H-ras and N-ras are dispensable for T-cell development and activation but critical for protective Th1 immunity

The small guanine nucleotide binding proteins of the Ras family, including in mammals the highly homologous H-ras, N-ras, and K-ras isoforms, are rapidly activated on ligation of the T-cell antigen receptor (TCR), but whether each isoform plays specific roles in T cells is largely unknown. Here, we show, with the use of mice specifically lacking H-ras or N-ras, that these isoforms are dispensable for thymocyte development and mature T-cell activation. By contrast, CD4⁺ T cells from Ras-deficient mice exhibited markedly decreased production of the Th1 signature cytokine IFN-γ early after TCR stimulation, concomitantly with impaired induction of the Th1-specific transcription factor T-bet. Accordingly, Ras-deficient mice failed to mount a protective Th1 response in vivo against the intracellular parasite Leishmania major, although they could be rendered resistant to infection if a Th1-biased milieu was provided during parasite challenge. Collectively, our data indicate that the TCR recruits distinct Ras isoforms for signal transduction in developing and mature T cells, thus providing a mechanism for differential signaling from the same surface receptor. Furthermore, we demonstrate for the first time that H-ras and N-ras act as critical controllers of Th1 responses, mostly by transmitting TCR signals for Th1 priming of CD4⁺ T cells.

N-ras couples antigen receptor signaling to Eomesodermin and to functional CD8+ T cell memory but not to effector differentiation

N-ras−/− CD8+ T cells have an intrinsic defect in Eomes expression resulting in impaired generation of protective memory cells that can be rescued by enforced Eomes expression.

Differential antibody binding to the surface αβTCR·CD3 complex of CD4+ and CD8+ T lymphocytes is conserved in mammals and associated with differential glycosylation

We have previously shown that the surface alphabeta T cell antigen receptor (TCR).CD3 complex borne by human CD4(+) and CD8(+) T lymphocytes can be distinguished using mAbs. Using two unrelated sets of antibodies, we have now extended this finding to the surface alphabetaTCR.CD3 of seven additional mammalian species (six non-human primates and the mouse). We have also produced data supporting that differential glycosylation of the two main T cell subsets is involved in the observed TCR.CD3 antibody-binding differences in humans. First, we show differential lectin binding to human CD4(+) versus CD8(+) T lymphocytes, particularly with galectin 7. Second, we show that certain lectins can compete differentially with CD3 mAb binding to human primary CD4(+) and CD8(+) T lymphocytes. Third, N-glycan disruption using swainsonine was shown to increase mAb binding to the alphabetaTCR.CD3. We conclude that the differential antibody binding to the surface alphabetaTCR.CD3 complex of primary CD4(+) and CD8(+) T lymphocytes is phylogenetically conserved and associated with differential glycosylation. The differences may be exploited for therapeutic purposes, such as T cell lineage-specific immunosuppression of graft rejection. Also, the impact of glycosylation on CD3 antibody binding requires a cautious interpretation of CD3 expression levels and T cell numbers in clinical diagnosis.

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 plasma C3 is essential for the development of memory B, but not T, lymphocytes

To the Editor: Primary C3 deficiency is an extremely rare autosomalrecessive disease, with fewer than 50 families described worldwide. Plasma and intracellular C3 are considered B-cell receptor (BCR) and T-cell receptor (TCR) costimulators, respectively, but their contribution to lymphocyte biology remains obscure, particularly in humans. Reduced plasma C3 can be caused not only by primary C3 deficiency, due to loss-of-function C3 mutations, but also by secondary C3 deficiency or C3 consumption, due to gain-of-function C3 mutations or due to mutations in C3 regulators such as complement Factor I (CFI). We reasoned that comparing Band T-cell differentiation and function in primary and secondary plasma C3 deficiency might help to understand the role of plasma and intracellular C3 in adaptive immunity. We report the immunological features of lymphocytes from 9 individuals with low plasma C3 belonging to 6 families, with mutations causing primary or secondary C3 deficiency and, in some cases, chronic kidney disease stages 1 to 3 (see Fig E1,A, and Tables E1 and E3 in this article’s Online Repository at www.jacionline.org).

Enrichment of the rare CD4+ γδ T-cell subset in patients with atypical CD3δ deficiency

demonstrate the novel finding that human ILC2s highly express the signaling lymphocyte activation molecule family member CD84, although this expression is not selective for ILC2s. Allergen challenge did not increase levels of CD84 expression on ILC2s or CD4 cells. We next assessed the percentage of CRTH21 ILC2s in the peripheral blood of cat-allergic subjects before and 4 hours after nasal cat allergen or diluent challenges (Fig 2). The baseline percentage of CRTH21 cells within the lineage-negative population was 10.7 6 1.9 and 12.0 6 1.3 at the diluent and cat allergen challenge visit, respectively (Fig 2, B). Four hours after diluent challenge, the percentage of CRTH21 cells did not change significantly (9.7 6 1.8) compared with time zero. However, after cat allergen challenge, the percentage of CRTH21 cells nearly doubled to 19.1 6 2.6 compared with baseline (P5 .05) and compared with diluent challenge at 4 hours (P < .05) (Fig 2, B). Thus, nasal cat allergen challenge induced an increased percentage of peripheral blood CRTH21 ILC2s when measured 4 hours after challenge. ILC2s produce large amounts of IL-5 and IL-13 in response to IL-25, IL-33, TSLP, and LTD4 and could initiate and/or propagate allergic airway inflammation. Our studies demonstrate that the percentage of CRTH21 ILC2s in the peripheral blood is rapidly increased (within 4 hours) after allergen challenge. Potential mechanisms for the increase in ILC2s in the peripheral blood may be due to enhanced recruitment of ILC2s from the bone marrow triggered by either humoral (cytokine, chemokine, or mediator production in the nose) and/or cellular mechanisms (cells released from the nasal mucosa trafficking to the bone marrow). The human ILC2 marker CRTH2 is the receptor for prostaglandin D2 (PGD2), a lipid mediator that has a known role in chemotaxis and activation of immune cells. Importantly, a previous study demonstrated that high levels of serum 9a,11b-PGF2, the major PGD2 metabolite, are induced within 5 minutes after airway allergen challenge, suggesting that PGD2 is rapidly available systemically for the recruitment of CRTH21 cells after allergen exposure. We have also recently determined that PGD2 induces chemotaxis of CRTH21 human blood ILC2s in vitro, suggesting that PGD2 may directly regulate the migration of human ILC2s into tissues. The role of increased peripheral blood ILC2 numbers after allergen challenge is unclear. One hypothesis is that greater ILC2 availability in the blood (within 4 hours after challenge) may result in greater numbers of cytokine-producing nasal mucosa ILC2s at later time points, but this would need to be investigated in future studies. Strategies to inhibit the recruitment of ILC2s in allergic individuals may reduce tissue TH2 cytokine levels that contribute to allergic inflammation.

Human Invariant Natural Killer T Cells Respond to Antigen-Presenting Cells Exposed to Lipids from Olea europaea Pollen

Background: Allergic sensitization might be influenced by the lipids present in allergens, which can be recognized by natural killer T (NKT) cells on antigen-presenting cells (APCs). The aim of this study was to analyze the effect of olive pollen lipids in human APCs, including monocytes as well as monocyte-derived macrophages (Mϕ) and dendritic cells (DCs). Methods: Lipids were extracted from olive (Olea europaea) pollen grains. Invariant (i)NKT cells, monocytes, Mϕ, and DCs were obtained from buffy coats of healthy blood donors, and their cell phenotype was determined by flow cytometry. iNKT cytotoxicity was measured using a lactate dehydrogenase assay. Gene expression of CD1A and CD1D was performed by RT-PCR, and the production of IL-6, IL-10, IL-12, and TNF-α cytokines by monocytes, Mϕ, and DCs was measured by ELISA. Results: Our results showed that monocytes and monocyte-derived Mϕ treated with olive pollen lipids strongly activate iNKT cells. We observed several phenotypic modifications in the APCs upon exposure to pollen-derived lipids. Both Mϕ and monocytes treated with olive pollen lipids showed an increase in CD1D gene expression, whereas upregulation of cell surface CD1d protein occurred only in Mϕ. Furthermore, DCs differentiated in the presence of human serum enhance their surface CD1d expression when exposed to olive pollen lipids. Finally, olive pollen lipids were able to stimulate the production of IL-6 but downregulated the production of lipopolysaccharide- induced IL-10 by Mϕ. Conclusions: Olive pollen lipids alter the phenotype of monocytes, Mϕ, and DCs, resulting in the activation of NKT cells, which have the potential to influence allergic immune responses.

Primary T-cell immunodeficiency with functional revertant somatic mosaicism in CD247

To the Editor: T lymphocytes detect antigens with the T-cell receptor (TCR) composed of a variable heterodimer (either ab or gd), 2 invariant heterodimers (CD3gε and CD3dε), and an invariant homodimer (CD247 or zz). Because of the crucial role of TCR signaling in thymic selection, mutations in TCR, CD3, or CD247 selectively impair T-cell development, albeit to different degrees: deficiency of CD3d or CD3ε, but not of CD3g or CD247, causes severe T-cell lymphopenia. Their clinical outcome is also disparate, because CD3g deficiency does not require urgent transplantation. Thus, TCR immunodeficiencies display a range of phenotypes and careful differential diagnosis is essential for appropriate therapy. We describe an infant born to consanguineous parents with early-onset chronic cytomegalovirus infection, severe immunodeficiency, and extremely low surface TCR levels. Her immunologic characterization at age 11 months is summarized in Table E1 in this article’s Online Repository at www.jacionline.