Multivalent structure of an αβT cell receptor

Whether there is one or multiple αβT cell antigen receptor (TCR) recognition modules in a given TCR/CD3 complex is a long-standing controversy in immunology. We show that T cells from transgenic mice that coexpress comparable amounts of two distinct TCRβ chains incorporate at least two αβTCRs in a single TCR/CD3 complex. Evidence for bispecific αβTCRs was obtained by immunoprecipitation and immunoblotting and confirmed on the surface of living cells both by fluorescence resonance energy transfer and comodulation assays by using antibodies specific for TCRβ-variable regions. Such (αβ)₂TCR/CD3 or higher-order complexes were evident in T cells studied either ex vivo or after expansion in vitro. T cell activation is thought by many, but not all, to require TCR cross-linking by its antigen/major histocompatibility complex ligand. The implications of a multivalent (αβ)₂TCR/CD3 complex stoichiometry for the ordered docking of specific antigen/major histocompatibility complex, CD4, or CD8 coreceptors and additional TCRs are discussed.     

T cell vaccination induces T cell receptor Vβ-specific Qa-1-restricted regulatory CD8+ T cells

Vaccination of mice with activated autoantigen-reactive CD4⁺ T cells (T cell vaccination, TCV) has been shown to induce protection from the subsequent induction of a variety of experimental autoimmune diseases, including experimental allergic encephalomyelitis (EAE). Although the mechanisms involved in TCV-mediated protection are not completely known, there is some evidence that TCV induces CD8⁺ regulatory T cells that are specific for pathogenic CD4⁺ T cells. Previously, we demonstrated that, after superantigen administration in vivo, CD8⁺ T cells emerge that preferentially lyse and regulate activated autologous CD4⁺ T cells in a T cell receptor (TCR) Vβ-specific manner. This TCR Vβ-specific regulation is not observed in β₂-microglobulin-deficient mice and is inhibited, in vitro, by antibody to Qa-1. We now show that similar Vβ8-specific Qa-1-restricted CD8⁺ T cells are also induced by TCV with activated CD4⁺ Vβ8⁺ T cells. These CD8⁺ T cells specifically lyse murine or human transfectants coexpressing Qa-1 and murine TCR Vβ8. Further, CD8⁺ T cell hybridoma clones generated from B10.PL mice vaccinated with a myelin basic protein-specific CD4⁺Vβ8⁺ T cell clone specifically recognize other CD4⁺ T cells and T cell tumors that express Vβ8 and the syngeneic Qa-1^a but not the allogeneic Qa-1^b molecule. Thus, Vβ-specific Qa-1-restricted CD8⁺ T cells are induced by activated CD4⁺ T cells. We suggest that these CD8⁺ T cells may function to specifically regulate activated CD4⁺ T cells during immune responses.     

Kinetics of T cell receptor β, γ, and δ rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44+CD25+ Pro-T thymocytes

We performed a comprehensive analysis of T cell receptor (TCR) γ rearrangements in T cell precursors of the mouse adult thymus. Using a sensitive quantitative PCR method, we show that TCRγ rearrangements are present in CD44⁺CD25⁺ Pro-T thymocytes much earlier than expected. TCRγ rearrangements increase significantly from the Pro-T to the CD44⁻CD25⁺ Pre-T cell transition, and follow different patterns depending on each Vγ gene segment, suggesting that ordered waves of TCRγ rearrangement exist in the adult mouse thymus as has been described in the fetal mouse thymus. Recombinations of TCRγ genes occur concurrently with TCRδ and D-Jβ rearrangements, but before Vβ gene assembly. Productive TCRγ rearrangements do not increase significantly before the Pre-T cell stage and are depleted in CD4⁺CD8⁺ double-positive cells from normal mice. In contrast, double-positive thymocytes from TCRδ−/− mice display random proportions of TCRγ rearranged alleles, supporting a role for functional TCRγ/δ rearrangements in the γδ divergence process.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

Failure of T cell homing, reduced CD4/CD8αα intraepithelial lymphocytes, and inflammation in the gut of vitamin D receptor KO mice

Specific pathogen-free IL-10 KO mice failed to develop inflammatory bowel disease (IBD), whereas IL-10/vitamin D receptor (VDR) double KO mice developed fulminating IBD. WT CD4 T cells inhibited experimental IBD, while VDR KO CD4 T cells failed to suppress IBD. VDR KO mice had normal numbers and functions of regulatory T cells. The percentages of IL-17- and IFN-γ-secreting T cells in the gut of mice reconstituted with WT and VDR KO CD4 T cells were also not different. Instead, there were twice as many CD8αα intraepithelial lymphocytes (IEL) in mice that were reconstituted with WT CD4 T cells than in mice reconstituted with VDR KO CD4 T cells. Furthermore, VDR KO mice had reduced numbers of CD8αα IEL, absent CD4/CD8αα populations, and as a result low IL-10 production in the IEL. The lack of CD8αα IEL was due in part to decreased CCR9 expression on T cells that resulted in the failure of the VDR KO T cells to home to the small intestine. We conclude that the VDR mediates T cell homing to the gut and as a result the VDR KO mouse has reduced numbers of CD8αα IEL with low levels of IL-10 leading to increased inflammatory response to the normally harmless commensal flora.     

Characterization of Adaptive-like γδ T Cells in Ugandan Infants during Primary Cytomegalovirus Infection

Gamma-delta (γδ) T cells are unconventional T cells that help control cytomegalovirus (CMV) infection in adults. γδ T cells develop early in gestation, and a fetal public γδ T cell receptor (TCR) clonotype is detected in congenital CMV infections. However, age-dependent γδ T cell responses to primary CMV infection are not well-understood. Flow cytometry and TCR sequencing was used to comprehensively characterize γδ T cell responses to CMV infection in a cohort of 32 infants followed prospectively from birth. Peripheral blood γδ T cell frequencies increased during infancy, and were higher among CMV-infected infants relative to uninfected. Clustering analyses revealed associations between CMV infection and activation marker expression on adaptive-like Vδ1 and Vδ3, but not innate-like Vγ9Vδ2 γδ T cell subsets. Frequencies of NKG2C⁺CD57⁺ γδ T cells were temporally associated with the quantity of CMV shed in saliva by infants with primary infection. The public γδ TCR clonotype was only detected in CMV-infected infants <120 days old and at lower frequencies than previously described in fetal infections. Our findings support the notion that CMV infection drives age-dependent expansions of specific γδ T cell populations, and provide insight for novel strategies to prevent CMV transmission and disease.     

Specific requirement for CD3ɛ in T cell development

T cell antigen receptor (TCR) and pre-TCR complexes are composed of clonotypic heterodimers in association with dimers of signal transducing invariant subunits (CD3γ, -δ, -, and ζ). The role of individual invariant subunits in T cell development has been investigated by generating gene-specific mutations in mice. Mutation of CD3γ, -δ, or ζ results in an incomplete block in development, characterized by reduced numbers of mature T cells that express low levels of TCR. In contrast, mature T cells are absent from CD3^−/− mice, and thymocyte development is arrested at the early CD4⁻CD8⁻ stage. Although these results suggest that CD3 is essential for pre-TCR and TCR expression/function, their interpretation is complicated by the fact that expression of the CD3γ and CD3δ genes also is reduced in CD3^−/− mice. Thus, it is unclear whether the phenotype of CD3^−/− mice reflects the collective effects of CD3γ, -δ, and - deficiency. By removing the selectable marker (PGK-NEO) from the targeted CD3 gene via Cre/loxP-mediated recombination, we generated mice that lack CD3 yet retain normal expression of the closely linked CD3γ and CD3δ genes. These (CD3^Δ/Δ) mice exhibited an early arrest in T cell development, similar to that of CD3^−/− mice. Moreover, the developmental defect could be rescued by expression of a CD3 transgene. These results identify an essential role for CD3 in T cell development not shared by the CD3γ, CD3δ, or ζ-family proteins and provide further evidence that PGK-NEO can influence the expression of genes in its proximity.     

αβ T cell receptor interactions with syngeneic and allogeneic ligands: Affinity measurements and crystallization

Cellular immunity is mediated by the interaction of an αβ T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have αβ TCRs that can recognize both self- and foreign peptide–MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive αβ T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired αβ heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2K^b-dEV8, H-2K^bm3-dEV8, H-2K^b-SIYR, and H-2L^d-p2Ca spans a range of affinities from K_d = 10⁻⁴ to 10⁻⁶M for the syngeneic (H-2K^b) and allogeneic (H-2K^bm3, H-2L^d) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the α and β chains. X-ray quality crystals of complexes of 2C with H-2K^b-dEV8, H-2K^bm3-dEV8 and H-2K^b-SIYR have been grown.     

CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages

It has been demonstrated that CD8⁺ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4⁺ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8⁺ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8⁺ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8⁺ T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8⁺ T-cell supernatants; and (iii) the CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8⁺ T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8⁺ T-cell-derived HIV-suppressor factors.     

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 in vivo, 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.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy arising from transformed immature T cell precursors (1). It represents ∼15% of pediatric and ∼25% of adult ALLs (2). Identification of subgroups with varied biological features, including overall relapse risk and responses to standard therapies, has allowed stratification of patients to the most appropriate therapeutic regimens that maximize efficacy, and has led to generally improved survival. However, prognosis remains poor for patients with treatment-refractory primary disease or relapse (3, 4). Clinical T-ALL can show inter- and intrapatient heterogeneity in the differentiation stage of tumor cells, implying that multiple pathways of cancer development exist (1, 5). Despite the heterogeneity, a unifying oncogenic network hub required for most or all T-ALL in humans and mice is hyperactivated (often mutated) NOTCH (6). Among the best understood causal drivers is developmentally early CD3 signaling at the pre-T cell antigen receptor (TCR)/γδTCR lineage bifurcation checkpoint, without a role for major histocompatibility complex (MHC)-based ligand (7–9). In contrast, a requirement for MHC and mature αβTCR to drive thymic leukemogenesis, resulting in mutant NOTCH-bearing tumors, has not been previously demonstrated.This makes sense based on the known relationship between T-ALL and T cell development. While there is overlap in NOTCH and developmentally early CD3 signals, cessation of NOTCH prior to MHC-restricted positive/negative selection signals mediated by αβTCR largely prevents simultaneous activity of the two receptors. Most conventional thymocytes rearrange first TCRβ and later TCRα loci in separate, ordered developmental stages. NOTCH signaling is required for early CD4⁻CD8⁻ double negative (DN) thymocyte development (10) while rearrangement of TCRβ and pre-TCR expression mediate β-selection, clonal expansion, and advancement to CD4⁺CD8⁺ double-positive (DP) stage (11). NOTCH signaling is then turned off (12), while DP thymocytes rearrange TCRα, express mature αβTCR, and test self-peptide-MHC reactivity in positive/negative selection (13).However, outside of this well-described sequence of events, a low-frequency, natural subset of DN thymocytes was once shown to rearrange and prematurely express the full αβTCR in wild-type mice (14). The cells were first detected in pre-Tα^−/− mice, where early TCRα replaced pre-Tα to provide β-selection signaling to generate DP cells. Conventional αβ T cell development potential was retained as proven in positive selection assays, but subsequent to the initial report little information on biological roles for these cells has followed. While pursuing developmental stages and signals in T-ALL leukemogenesis, we found that early αβTCR-expressing DN (EADN) cells can be generated in mouse and human thymus at a similar rate, and they are susceptible to T-ALL transformation in both species. We present a mouse model in which EADN oncogenesis requires MHC to drive development of T-ALL bearing NOTCH-mutations, highlighting a novel developmental state with unique signaling rules for a cancer phenotype that appears to be represented in the clinical, human disease spectrum.     

In differentiated CD4+ T Cells, interleukin 4 production is cytokine-autonomous, whereas interferon γ production is cytokine-dependent

CD4⁺ T cells from T cell receptor transgenic mice that have been vigorously primed to be interleukin (IL)-4 producers (T_H2 cells) are capable of producing IL-4 even if restimulated in the absence of IL-4 and in the presence of IL-12. T cells vigorously primed in the absence of IL-4 (T_H1 cells) fail to produce IL-4 even if restimulated under conditions that would cause a naive T cell to produce IL-4. In contrast, interferon γ (IFN-γ) production is highly cytokine-regulated. T cells primed in the presence of IL-4 develop into IFN-γ producers if IFN-γ is included in the priming culture and if the cells are challenged in the presence of IL-12, presumably reflecting the role of IFN-γ in inducing responsiveness to IL-12. Cells primed in the absence of IL-4 become highly responsive to IL-12 if IFN-γ is included in the priming culture, and these cells are excellent IFN-γ producers upon challenge; IL-12 considerably enhances their production of IFN-γ. If cells are primed in the absence of IL-4 and IFN-γ, they show very weak responsiveness to IL-12 as determined by STAT-4 activation. However, these cells acquire IL-12 responsiveness if cultured with IFN-γ for a period as short as 4 hr. Thereafter, they produce large amounts of IFN-γ upon challenge with antigen in the presence of IL-12. These results indicate that in primed CD4⁺ T cells, IL-4 production is largely cytokine-autonomous, whereas IFN-γ production is highly cytokine-regulated.     

Rapid deletion of rearranged T cell antigen receptor (TCR) Vα-Jα segment by secondary rearrangement in the thymus: Role of continuous rearrangement of TCR α chain gene and positive selection in the T cell repertoire formation

A rearranged T cell receptor (TCR) Vα and Jα gene from a cytochrome c-specific T cell hybridoma was introduced into the genomic Jα region. The introduced TCR α chain gene is expressed in a majority of CD3 positive and CD4 CD8 double-negative immature thymocytes. However, only a few percent of the double-positive and single-positive thymocytes express this TCR α chain. This decrease is caused by a rearrangement of TCR α chain locus, which deletes the introduced TCR gene. Analysis of the mice carrying the introduced TCR α chain and the transgenic TCR β chain from the original cytochrome c-specific T cell hybridoma revealed that positive selection efficiently rescues double-positive thymocytes from the loss of the introduced TCR α chain gene. In the mice with negatively selecting conditions, T cells expressing the introduced TCR αβ chains were deleted at the double-positive stage. However, a large number of thymocytes escape negative selection by using an endogenous TCR α chain created by secondary rearrangement maintaining normal thymocyte development. These results suggest that secondary rearrangements of the TCR α chain gene play an important role in the formation of the T cell repertoire.     

Revisiting the Role of γδ T Cells in Anti-CMV Immune Response after Transplantation

Gamma delta (γδ) T cells form an unconventional subset of T lymphocytes that express a T cell receptor (TCR) consisting of γ and δ chains. Unlike conventional αβ T cells, γδ T cells share the immune signature of both the innate and the adaptive immunity. These features allow γδ T cells to act in front-line defense against infections and tumors, rendering them an attractive target for immunotherapy. The role of γδ T cells in the immune response to cytomegalovirus (CMV) has been the focus of intense research for several years, particularly in the context of transplantation, as CMV reactivation remains a major cause of transplant-related morbidity and mortality. Therefore, a better understanding of the mechanisms that underlie CMV immune responses could enable the design of novel γδ T cell-based therapeutic approaches. In this regard, the advent of next-generation sequencing (NGS) and single-cell TCR sequencing have allowed in-depth characterization of CMV-induced TCR repertoire changes. In this review, we try to shed light on recent findings addressing the adaptive role of γδ T cells in CMV immunosurveillance and revisit CMV-induced TCR reshaping in the era of NGS. Finally, we will demonstrate the favorable and unfavorable effects of CMV reactive γδ T cells post-transplantation.     

β cell apoptosis in T cell-mediated autoimmune diabetes

Insulin-dependent diabetes mellitus results from T cell-mediated destruction of insulin-producing, pancreatic islet β cells. How this destruction takes place has remained elusive—largely due to the slow kinetics of disease progression. By crossing a transgenic mouse carrying a β cell-specific T cell receptor onto the NOD.scid background, we produced a simplified but robust and accelerated model of diabetes. This mouse produces CD4⁺ T cells bearing transgenic T cell receptor but is devoid of CD8⁺ T cells and B cells. More importantly, this mouse develops a rapid diabetes, which has allowed us to record and quantify β cell death. We have determined that β cells within the inflamed islets die by apoptosis.     

Primary CD8+ cells from HIV-infected individuals can suppress productive infection of macrophages independent of β-chemokines

The productive infection of human monocyte-derived macrophages (M) by HIV was suppressed by primary CD8⁺ cells from asymptomatic HIV-infected individuals. This anti-HIV response was noncytotoxic; removal of the CD8⁺ cells from the infected M leads to virus production. CD8⁺ cells inhibited HIV replication when separated from the infected M by a transwell filter insert, indicating a diffusible factor made by the CD8⁺ cells suppressed productive infection of M. Three β-chemokines, which can be secreted by activated CD8⁺ cells, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1α and MIP-1β prevented HIV replication in the M cultures. In addition, incubation of acutely infected M with a mixture of neutralizing antibodies to RANTES, MIP-1α, and MIP-1β enhanced virus replication. Nevertheless, neutralization of β-chemokines with specific antibodies did not abolish the suppression by CD8⁺ cells of HIV replication in M. Thus, even though β-chemokines decrease HIV replication in M, these cytokines are not responsible for the ability of CD8⁺ cells to inhibit HIV production in these cells.     

An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus

T cell receptor (TCR) α and δ gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3′ of TCR δ gene segments and 5′ of TCR α joining gene segments within this locus. BEAD-1 blocked the ability of the TCR δ enhancer (E_δ) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR α/δ locus into distinct regulatory domains controlled by E_δ and the TCR α enhancer, and that it prevents E_δ from opening the chromatin of the TCR α joining gene segments for VDJ recombination at an early stage of T cell development.     

T cell functions in granulocyte/macrophage colony-stimulating factor deficient mice

Immunological functions were analyzed in mice lacking granulocyte/macrophage colony-stimulating factor (GM-CSF). The response of splenic T cells to allo-antigens, anti-mouse CD3 mAb, interleukin 2 (IL-2), or concanavalin A was comparable in GM-CSF +/+ and GM-CSF −/− mice. To investigate the responses of CD8⁺ and CD4⁺ T cells against exogenous antigens, mice were immunized with ovalbumin peptide or with keyhole limpet hemocyanin (KLH). Cytotoxic CD8⁺ T cells with specificity for ovalbumin peptide could not be induced in GM-CSF −/− mice. After immunization with KLH, there was a delay in IgG generation, particularly IgG2a, in GM-CSF −/− mice. Purified CD4⁺ T cells from GM-CSF −/− mice immunized with KLH showed impaired proliferative responses and produced low amounts of interferon-γ (IFN-γ) and IL-4 when KLH-pulsed B cells or spleen cells were used as antigen presenting cells (APC). When enriched dendritic cells (DC) were used as APC, CD4⁺ T cells from GM-CSF −/− mice proliferated as well as those from GM-CSF +/+ mice and produced high amounts of IFN-γ and IL-4. To analyze the rescue effect of DC on CD4⁺ T cells, supernatants from (i) CD4⁺ T cells cultured with KLH-pulsed DC or (ii) DC cultured with recombinant GM-CSF were transferred to cultures of CD4⁺ T cells and KLH-pulsed spleen cells from GM-CSF −/− mice. Supernatants from both DC sources contained a factor or factors that restored proliferative responses and IFN-γ production of CD4⁺ T cells from GM-CSF −/− mice.     

Anti-HIV-1 and chemotactic activities of human stromal cell-derived factor 1α (SDF-1α) and SDF-1β are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage

CD26 is a leukocyte-activation antigen that is expressed on T lymphocytes and macrophages and possesses dipeptidyl peptidase IV (DPPIV) activity, whose natural substrates have not been identified yet. CXC chemokines, stromal cell-derived factor 1α (SDF-1α) and 1β (SDF-1β), sharing the receptor CXCR-4, are highly efficacious chemoattractants for resting lymphocytes and CD34⁺ progenitor cells, and they efficiently block the CXCR-4-mediated entry into cells of T cell line tropic strains of HIV type 1 (HIV-1). Here we show that both the chemotactic and antiviral activities of these chemokines are abrogated by DPPIV-mediated specific removal of the N-terminal dipeptide, not only when the chemokines are produced in transformed mouse L cell line to express human CD26 but also when they were exposed to a human T cell line (H9) physiologically expressing CD26. Mutagenesis of SDF-1α confirmed the critical requirement of the N-terminal dipeptide for its chemotactic and antiviral activities. These data suggest that CD26-mediated cleavage of SDF-1α and SDF-1β likely occurs in human bodies and promotes HIV-1 replication and disease progression. They may also explain why memory function of CD4⁺ cells is preferentially lost in HIV-1 infection. Furthermore, CD26 would modulate various other biological processes in which SDF-1α and SDF-1β are involved.     

A critical role for neutralizing-antibody-producing B cells, CD4+ T cells, and interferons in persistent and acute infections of mice with lymphocytic choriomeningitis virus: Implications for adoptive immunotherapy of virus carriers

This study demonstrates that neutralizing-antibody-producing B cells, CD4⁺ T cells, and interferons (IFNs) are of key importance in virus control both in adoptive immunotherapy of persistent infection and in the late phase of acute infection with the WE strain of lymphocytic choriomeningitis virus (LCMV). We report the following results. (i) Clearance of LCMV-WE from C57BL/6 carrier mice by adoptive transfer of memory spleen cells requires B cells and CD4⁺ T cells but not necessarily CD8⁺ T cells. (ii) At the doses examined, CD8⁺ T cells contribute to the initial reduction of viral titers but are alone not sufficient to clear the virus because they are exhausted. (iii) In the presence of functional IFN-γ, virus clearance correlates well with the generation of neutralizing antibodies in the treated carrier mice. (iv) In the absence of receptors for IFN-γ, virus clearance is not achieved. (v) Adoptive immunotherapy of mice persistently infected with a distinct virus isolate, LCMV-Armstrong, revealed only low levels of neutralizing antibodies; in this case, CD8⁺ T cells were needed for virus clearance in addition to B and CD4⁺ T cells. (vi) After low dose infection of C57BL/6 mice with LCMV-WE, virus is eliminated below detectable levels by CD8⁺ T cells, but long-term (>2 months) virus control is usually not achieved in the absence of B cells or CD4⁺ T cells; reappearance of the virus is paralleled either by exhaustion of virus-specific cytotoxic T lymphocytes or lethal immunopathology. These findings are of importance for adoptive immunotherapy strategies against persistent virus infections in humans.