Biomechanical and metabolic aspects of backward (and forward) running on uphill gradients: another clue towards an almost inelastic rebound.

PURPOSE: On level, the metabolic cost (C) of backward running is higher than forward running probably due to a lower elastic energy recoil. On positive gradient, the ability to store and release elastic energy is impaired in forward running. We studied running on level and on gradient to test the hypothesis that the higher metabolic cost and lower efficiency in backward than forward running was due to the impairment in the elastic energy utilisation. METHODS: Eight subjects ran forward and backward on a treadmill on level and on gradient (from 0 to + 25%, with 5% step). The mechanical work, computed from kinematic data, C and efficiency (the ratio between total mechanical work and C) were calculated in each condition. RESULTS: Backward running C was higher than forward running at each condition (on average + 35%) and increased linearly with gradient. Total mechanical work was higher in forward running only at the steepest gradients, thus efficiency was lower in backward running at each gradient. CONCLUSION: Efficiency decreased by increasing gradient in both running modalities highlighting the impairment in the elastic contribution on positive gradient. The lower efficiency values calculated in backward running in all conditions pointed out that backward running was performed with an almost inelastic rebound; thus, muscles performed most of the mechanical work with a high metabolic cost. These new backward running C data permit, by applying the recently introduced 'equivalent slope' concept for running acceleration, to obtain the predictive equation of metabolic power during level backward running acceleration.

Clinical correlates of augmentation/combination treatment strategies in major depressive disorder.

OBJECTIVE: This multicenter, multinational, cross-sectional study aimed to investigate clinical characteristics and treatment outcomes associated with augmentation/combination treatment strategies in major depressive disorder (MDD). METHOD: Sociodemographic, clinical, and treatment features of 1410 adult MDD patients were compared between MDD patients treated with monotherapy and augmentation/combination medication using descriptive statistics, analyses of covariance (ancova), and Spearman's correlation analyses. RESULTS: 60.64% of all participants received augmentation and/or combination strategies with a mean number of 2.18 ± 1.22 simultaneously prescribed psychiatric drugs. We found male gender, older age, Caucasian descent, higher weight, low educational status, absence of occupation, psychotic symptoms, melancholic and atypical features, suicide risk, in-patient treatment, longer duration of hospitalization, some psychiatric comorbidities (panic disorder, agoraphobia, obsessive-compulsive disorder, and bulimia nervosa), comorbid somatic comorbidity in general and concurrent hypertension, thyroid dysfunction, diabetes, and heart disease in particular, higher current and retrospective Montgomery and Åsberg Depression Rating Scale total scores, treatment resistance, and higher antidepressant dosing to be significantly associated with augmentation/combination treatment. These findings were corroborated when examining the number of concurrently administered psychiatric drugs in the statistical analyses. CONCLUSION: Our findings suggest a clear association between augmentation/combination strategies and treatment-resistant/difficult-to-treat MDD conditions characterized by severe symptomatology and high amount of psychiatric and somatic comorbidities.

Direct incorporation of the NKT-cell activator α-galactosylceramide into a recombinant Listeria monocytogenes improves breast cancer vaccine efficacy.

BACKGROUND: Immune suppression in the tumour microenvironment remains a major limitation to successful immunotherapy of cancer. In the current study, we analysed whether the natural killer T cell-activating glycolipid α-galactosylceramide could overcome immune suppression and improve vaccination against metastatic breast cancer. METHODS: Mice with metastatic breast cancer (4T1 model) were therapeutically treated with a Listeria monocytogenes-based vaccine expressing tumour-associated antigen Mage-b followed by α-galactosylceramide as separate agents, or as a complex of α-galactosylceramide stably incorporated into Listeria-Mage-b. Effects on metastases, tumour weight, toxicity and immune responses were determined. RESULTS: Sequential treatments of mice with established 4T1 breast carcinomas using Listeria-Mage-b followed by α-galactosylceramide as a separate agent was highly effective at reducing metastases, but was accompanied by severe liver toxicity. In contrast, combined therapy using Listeria-Mage-b modified by incorporation of α-galactosylceramide resulted in nearly complete elimination of metastases without toxicity. This was associated with a significant increase in the percentage of natural killer T cells in the spleen, and an increase in natural killer cell activity and in T cell responses to Mage-b. CONCLUSIONS: Our results suggest that direct incorporation of α-galactosylceramide into a live bacterial vaccine vector is a promising non-toxic new approach for the treatment of metastatic breast cancer.

T helper type 2-polarized invariant natural killer T cells reduce disease severity in acute intra-abdominal sepsis.

Sepsis is characterized by a severe systemic inflammatory response to infection that is associated with high morbidity and mortality despite optimal care. Invariant natural killer T (iNK T) cells are potent regulatory lymphocytes that can produce pro- and/or anti-inflammatory cytokines, thus shaping the course and nature of immune responses; however, little is known about their role in sepsis. We demonstrate here that patients with sepsis/severe sepsis have significantly elevated proportions of iNK T cells in their peripheral blood (as a percentage of their circulating T cells) compared to non-septic patients. We therefore investigated the role of iNK T cells in a mouse model of intra-abdominal sepsis (IAS). Our data show that iNK T cells are pathogenic in IAS, and that T helper type 2 (Th2) polarization of iNK T cells using the synthetic glycolipid OCH significantly reduces mortality from IAS. This reduction in mortality is associated with the systemic elevation of the anti-inflammatory cytokine interleukin (IL)-13 and reduction of several proinflammatory cytokines within the spleen, notably interleukin (IL)-17. Finally, we show that treatment of sepsis with OCH in mice is accompanied by significantly reduced apoptosis of splenic T and B lymphocytes and macrophages, but not natural killer cells. We propose that modulation of iNK T cell responses towards a Th2 phenotype may be an effective therapeutic strategy in early sepsis.

Salivary steroid hormone response to whole-body cryotherapy in elite rugby players.

Saliva represents a low stress, not-invasively collected matrix that allows steroid hormone monitoring in athletes by reflecting type, intensity and duration of exercise. Whole body cryotherapy (WBC) consists of short whole-body exposures to extremely cold air (-110° to -140°C) which, despite being initially used to treat inflammatory diseases, is currently acquiring increasing popularity in sports medicine. Cryostimulation practice is now widely accepted as an effective treatment to accelerate muscle recovery in rugby players. The aim of this work was to study the changes of steroid hormones in saliva of rugby players after both 2 and 14 consecutive WBC sessions, in order to investigate the effects of the treatment on their salivary steroid hormonal profile. Twenty-five professional rugby players, belonging to the Italian National Team, underwent a 7-day cryotherapy protocol consisting of 2 daily sessions. Saliva samples were taken in the morning prior to the start of the WBC, in the evening after the end of the second WBC, and in the morning of the day after the last WBC session. The samples were analyzed for cortisol, DHEA, testosterone and estradiol using competitive enzyme-linked immunosorbent assays. Cortisol and DHEA showed a reduction already after the 2 WBC sessions of the first day; after 14 consecutive WBC sessions cortisol, DHEA, and estradiol levels decreased, while testosterone increased as did the testosterone to cortisol ratio. These results were confirmed by the fact that the majority of subjects showed variations exceeding the critical difference (CD). In conclusion, we found that WBC acutely affects the salivary steroid hormone profile, and the results are evident already after only one twice-daily session. Most significantly, after one-week of consecutive twice-daily WBC sessions, all the hormones were modified. This is the first experimental report that links changes in the hormonal asset to WBC.

Stimulation of a shorter duration in the state of anergy by an invariant natural killer T cell agonist enhances its efficiency of protection from type 1 diabetes.

We have reported previously that treatment of non-obese diabetic (NOD) mice with the invariant natural killer T (iNK T) cell agonist α-galactosylceramide C26:0 (α-GalCer) or its T helper type 2 (Th2)-biasing derivative α-GalCer C20:2 (C20:2) protects against type 1 diabetes (T1D), with C20:2 yielding greater protection. After an initial response to α-GalCer, iNK T cells become anergic upon restimulation. While such anergic iNK T cells can induce tolerogenic dendritic cells (DCs) that mediate protection from T1D, chronic administration of α-GalCer also results in long-lasting anergy accompanied by significantly reduced iNK T cell frequencies, which raises concerns about its long-term therapeutic use. In this study, our objective was to understand more clearly the roles of anergy and induction of tolerogenic DCs in iNK T cell-mediated protection from T1D and to circumvent potential complications associated with α-GalCer. We demonstrate that NOD iNK T cells activated during multi-dose (MD) treatment in vivo with C20:2 enter into and exit from anergy more rapidly than after activation by α-GalCer. Importantly, this shorter duration of iNK T cells in the anergic state promotes the more rapid induction of tolerogenic DCs and reduced iNK T cell death, and enables C20:2 stimulated iNK T cells to elicit enhanced protection from T1D. Our findings further that suggest C20:2 is a more effective therapeutic drug than α-GalCer for protection from T1D. Moreover, the characteristics of C20:2 provide a basis of selection of next-generation iNK T cell agonists for the prevention of T1D.

CD1d-restricted recognition of synthetic glycolipid antigens by human natural killer T cells.

A conserved subset of mature circulating T cells in humans expresses an invariant Valpha24-JalphaQ T cell receptor (TCR)-alpha chain rearrangement and several natural killer (NK) locus-encoded C-type lectins. These human T cells appear to be precise homologues of the subset of NK1.1(+) TCR-alpha/beta+ T cells, often referred to as NK T cells, which was initially identified in mice. Here we show that human NK T cell clones are strongly and specifically activated by the same synthetic glycolipid antigens as have been shown recently to stimulate murine NK T cells. Responses of human NK T cells to these synthetic glycolipids, consisting of certain alpha-anomeric sugars conjugated to an acylated phytosphingosine base, required presentation by antigen-presenting cells expressing the major histocompatibility complex class I-like CD1d protein. Presentation of synthetic glycolipid antigens to human NK T cells required internalization of the glycolipids by the antigen-presenting cell and normal endosomal targeting of CD1d. Recognition of these compounds by human NK T cells triggered proliferation, cytokine release, and cytotoxic activity. These results demonstrate a striking parallel in the specificity of NK T cells in humans and mice, thus providing further insight into the potential mechanisms of immune recognition by NK T cells and the immunological function of this unique T cell subset.

Requirements for CD1d recognition by human invariant Valpha24+ CD4-CD8- T cells.

A subset of human CD4-CD8- T cells that expresses an invariant Valpha24-JalphaQ T cell receptor (TCR)-alpha chain, paired predominantly with Vbeta11, has been identified. A series of these Valpha24 Vbeta11 clones were shown to have TCR-beta CDR3 diversity and express the natural killer (NK) locus-encoded C-type lectins NKR-P1A, CD94, and CD69. However, in contrast to NK cells, they did not express killer inhibitory receptors, CD16, CD56, or CD57. All invariant Valpha24(+) clones recognized the MHC class I-like CD16 molecule and discriminated between CD1d and other closely related human CD1 proteins, indicating that recognition was TCR-mediated. Recognition was not dependent upon an endosomal targeting motif in the cytoplasmic tail of CD1d. Upon activation by anti-CD3 or CD1d, the clones produced both Th1 and Th2 cytokines. These results demonstrate that human invariant Valpha24+ CD4-CD8- T cells, and presumably the homologous murine NK1+ T cell population, are CD1d reactive and functionally distinct from NK cells. The conservation of this cell population and of the CD1d ligand across species indicates an important immunological function.

Both a monoclonal antibody and antisera specific for determinants unique to individual cloned helper T cell lines can substitute for antigen and antigen-presenting cells in the activation of T cells.

Two antisera and a monoclonal antibody raised in BALB.K mice against cloned, major histocompatibility complex (MHC)-restricted, antigen-specific helper T cell lines are described. These antibodies are specific for individual cloned T cell lines and are potent inducers of T cell proliferation. The induction of T cell proliferation by these antibodies requires the presence of an adherent accessory cell. There is no H-2 restriction between this accessory cell and the cloned T cell, nor is this antibody-induced proliferation blocked by a monoclonal anti-Fc receptor antibody. The requirement for an accessory cell, however, is eliminated in the presence of an IL-1- or IL-2-rich supernatant. Thus this system allows the analysis of helper T cell activation with only a single cell type present. Anti-T cell sera also induce T cell-dependent B cell proliferation and immunoglobulin secretion. The induction of T cell-dependent B cell activation by these sera does not require H-2-matched T cells and B cells. The specificity of these antibodies and their ability to stimulate cloned helper T cells in the absence of antigen and antigen-presenting cells strongly suggest that these antibodies are directed against antigen and/or Ia recognition sites on the T cell.

CD161 (NKR-P1A) costimulation of CD1d-dependent activation of human T cells expressing invariant V alpha 24 J alpha Q T cell receptor alpha chains.

A population of human T cells expressing an invariant V alpha 24 J alpha Q T cell antigen receptor (TCR) alpha chain and high levels of CD161 (NKR-P1A) appears to play an immunoregulatory role through production of both T helper (Th) type 1 and Th2 cytokines. Unlike other CD161(+) T cells, the major histocompatibility complex-like nonpolymorphic CD1d molecule is the target for the TCR expressed by these T cells (V alpha 24(invt) T cells) and by the homologous murine NK1 (NKR-P1C)+ T cell population. In this report, CD161 was shown to act as a specific costimulatory molecule for TCR-mediated proliferation and cytokine secretion by V alpha 24(invt) T cells. However, in contrast to results in the mouse, ligation of CD161 in the absence of TCR stimulation did not result in V alpha 24(invt) T cell activation, and costimulation through CD161 did not cause polarization of the cytokine secretion pattern. CD161 monoclonal antibodies specifically inhibited V alpha 24(invt) T cell proliferation and cytokine secretion in response to CD1d+ target cells, demonstrating a physiological accessory molecule function for CD161. However, CD1d-restricted target cell lysis by activated V alpha 24(invt) T cells, which involved a granule-mediated exocytotic mechanism, was CD161-independent. In further contrast to the mouse, the signaling pathway involved in V alpha 24(invt) T cell costimulation through CD161 did not appear to involve stable association with tyrosine kinase p56(Lck). These results demonstrate a role for CD161 as a novel costimulatory molecule for TCR-mediated recognition of CD1d by human V alpha 24(invt) T cells.

Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain.

CD4-CD8- (double negative [DN]) alpha/beta T cells are a largely uncharacterized subpopulation of unknown function. To investigate whether these cells are selected to recognize particular antigens or antigen-presenting molecules, DN alpha/beta T cells were purified from the peripheral blood of five normal donors and their T cell receptor (TCR) alpha and beta chains were examined. Random cloning of TCR alpha chains by single-sided polymerase chain reaction (PCR) amplification identified an invariant rearrangement between V alpha 24 and J alpha Q, with no N region diversity, which was expressed preferentially by DN alpha/beta T cells from all donors. Random cloning also identified a precise V alpha 7.2-J alpha (IGRJa14) rearrangement, with two variable amino acids encoded in the V-J junction, which was enriched in the DN alpha/beta T cell preparations from some, but not all, donors. Analysis of TCR beta chains by quantitative PCR amplification demonstrated that the expression of four V beta gene families, V beta 2, 8, 11, and 13, was markedly increased in these DN alpha/beta T cell preparations. The expression of particular TCRs by DN alpha/beta T cells from multiple donors indicates that these cells, or at least a subpopulation of cells with this phenotype, recognize a limited spectrum of antigens and suggests that they may use nonpolymorphic antigen-presenting molecules.

A pathway of costimulation that prevents anergy in CD28- T cells: B7-independent costimulation of CD1-restricted T cells.

A class of molecules that is expressed on antigen presenting cells, exemplified by CD80 (B7), has been found to provide a necessary costimulatory signal for T cell activation and proliferation. CD28 and CTLA4 are the B7 counterreceptors and are expressed on the majority of human CD4+ T cells and many CD8+ T cells. The signal these molecules mediate is distinguished from other costimulatory signals by the finding that T cell recognition of antigen results in a prolonged state of T cell unresponsiveness or anergy, unless these costimulatory molecules are engaged. However, nearly half of the CD8+ and CD4-CD8- T cells lack CD28, and the costimulatory signals required for the activation of such cells are unknown. To understand the pathways of activation used by CD28- T cells, we have examined the costimulatory requirements of antigen-specific CD4-CD8- TCR(+)-alpha/beta circulating T cells that lack the expression of CD28. We have characterized two T cell lines, DN1 and DN6, that recognize a mycobacterial antigen, and are restricted not by major histocompatibility complex class I or II, but by CD1b or CD1c, two members of a family of major histocompatibility complex-related molecules that have been recently implicated in a distinct pathway for antigen presentation. Comparison of antigen-specific cytolytic responses of the DN1 and DN6 T cell lines against antigen-pulsed CD1+ monocytes or CD1+ B lymphoblastoid cell lines (B-LCL) demonstrated that these T cells recognized antigen presented by both types of cells. However, T cell proliferation occurred only when antigen was presented by CD1+ monocytes, indicating that the CD1+ monocytes expressed a costimulatory molecule that the B-LCL transfectants lacked. This hypothesis was confirmed by demonstrating that the T cells became anergic when incubated with the CD1(+)-transfected B-LCL in the presence of antigen, but not in the absence of antigen. The required costimulatory signal occurred by a CD28-independent mechanism since both the CD1+ monocytes and CD1+ B-LCL transfectants expressed B7-1 and B7-2, and DN1 and DN6 lacked surface expression of CD28. We propose that these data define a previously unrecognized pathway of costimulation for T cells distinct from that involving CD28 and its counterreceptors. We suggest that this B7-independent pathway plays a crucial role in the activation and maintenance of tolerance of at least a subset of CD28- T cells.

Human CD1b and CD1c isoforms survey different intracellular compartments for the presentation of microbial lipid antigens.

CD1b and CD1c are antigen-presenting molecules that mediate recognition of bacterial lipids by T cells, but it is currently not known whether these two molecules are redundant or are specialized to perform different immunological functions. Here, we show that the distribution of CD1c in human dendritic cells was characterized by a high ratio of cell surface to intracellular molecules, whereas CD1b showed a reciprocal pattern of distribution. In contrast to the accumulation of CD1b in lysosomal major histocompatibility complex class II compartments, intracellular CD1c molecules accumulated in other endocytic compartments, most likely early and late endosomes. Deletion of the cytoplasmic tail of CD1c, containing a tyrosine-based internalization motif, abolished most of its intracellular localization. Functional studies using T cells specific for defined lipid antigens revealed that in contrast to CD1b-mediated antigen presentation, antigen presentation by CD1c was resistant to drugs inhibiting endosomal acidification and was independent of endosomal localization of CD1c. Taken together, these results support the hypothesis that CD1b and CD1c are specialized to survey the lipid content of different intracellular compartments.

CD1b-mediated T cell recognition of a glycolipid antigen generated from mycobacterial lipid and host carbohydrate during infection.

T cells recognize microbial glycolipids presented by CD1 proteins, but there is no information regarding the generation of natural glycolipid antigens within infected tissues. Therefore, we determined the molecular basis of CD1b-restricted T cell recognition of mycobacterial glycosylated mycolates, including those produced during tissue infection in vivo. Transfection of the T cell receptor (TCR) alpha and beta chains from a glucose monomycolate (GMM)-specific T cell line reconstituted GMM recognition in TCR-deficient T lymphoblastoma cells. This TCR-mediated response was highly specific for natural mycobacterial glucose-6-O-(2R, 3R) monomycolate, including the precise structure of the glucose moiety, the stereochemistry of the mycolate lipid, and the linkage between the carbohydrate and the lipid. Mycobacterial production of antigenic GMM absolutely required a nonmycobacterial source of glucose that could be supplied by adding glucose to media at concentrations found in mammalian tissues or by infecting tissue in vivo. These results indicate that mycobacteria synthesized antigenic GMM by coupling mycobacterial mycolates to host-derived glucose. Specific T cell recognition of an epitope formed by interaction of host and pathogen biosynthetic pathways provides a mechanism for immune response to those pathogenic mycobacteria that have productively infected tissues, as distinguished from ubiquitous, but innocuous, environmental mycobacteria.

Distinct cytokine profiles of neonatal natural killer T cells after expansion with subsets of dendritic cells.

Natural killer T (NKT) cells are a highly conserved subset of T cells that have been shown to play a critical role in suppressing T helper cell type 1-mediated autoimmune diseases and graft versus host disease in an interleukin (IL)-4-dependent manner. Thus, it is important to understand how the development of IL-4- versus interferon (IFN)-gamma-producing NKT cells is regulated. Here, we show that NKT cells from adult blood and those from cord blood undergo massive expansion in cell numbers (500-70,000-fold) during a 4-wk culture with IL-2, IL-7, phytohemagglutinin, anti-CD3, and anti-CD28 mAbs. Unlike adult NKT cells that preferentially produce both IL-4 and IFN-gamma, neonatal NKT cells preferentially produce IL-4 after polyclonal activation. Addition of type 2 dendritic cells (DC2) enhances the development of neonatal NKT cells into IL-4(+)IFN-gamma(-) NKT2 cells, whereas addition of type 1 dendritic cells (DC1) induces polarization towards IL-4(-)IFN-gamma(+) NKT1 cells. Adult NKT cells display limited plasticity for polarization induced by DC1 or DC2. Thus, newly generated NKT cells may possess the potent ability to develop into IL-4(+)IFN-gamma(-) NKT2 cells in response to appropriate stimuli and may thereafter acquire the tendency to produce both IL-4 and IFN-gamma.

Human lymphocytes bearing T cell receptor gamma/delta are phenotypically diverse and evenly distributed throughout the lymphoid system.

A direct quantitative and phenotypic cytofluorographic analysis of TCR-gamma/delta+ lymphocytes as well as an immunohistologic study of their tissue distribution and microanatomy was made possible by the availability of two mAbs (anti-TCR-delta 1 and anti-C gamma M1) specific for framework determinants on human TCR gamma and delta chains, respectively. TCR-gamma/delta+ lymphocytes, ranging between greater than 0.5 and 16% of CD3+ cells, were found in fetal and postnatal thymus, fetal and adult peripheral lymphoid organs, and adult peripheral blood. While TCR-gamma/delta+ lymphocytes comprised a small subpopulation of T cells (mean, approximately 4%) occasionally greater than 10-16% of CD3+ cells expressed TCR-gamma/delta. Virtually all TCR-gamma/delta+ thymocytes/lymphocytes expressed CD7, CD2, and CD5 but were heterogeneous with respect to their expression of CD1, CD4, CD8, CD28, CD11b, CD16, and Leu-7. Human TCR-gamma/delta+ cells populate both organized lymphoid tissues (thymus, tonsil, lymphnode, and spleen) as well as the gut- and skin-associated lymphoid systems at similar frequencies without obvious tropism for epithelial microenvironments. TCR-gamma/delta+ lymphocytes tend to be located within a given organ wherever TCR-alpha/beta+ lymphocytes are found. This study shows that TCR-gamma/delta+ lymphocytes constitute a small but numerically important, phenotypically diverse T cell population distributed throughout the body. These results support the concept that TCR-gamma/delta+ cells comprise a distinct, functionally heterogeneous, mature T cell sublineage that may substantially broaden the T cell repertoire at all immunologically relevant sites.

Evidence for extrathymic changes in the T cell receptor gamma/delta repertoire.

The germline repertoire of variable genes for the TCR-gamma/delta is limited. This, together with the availability of several V delta-specific and a C delta-specific mAbs, has made it possible to assess differences in the TCR-gamma/delta repertoire in man. TCR-gamma/delta cells expressing particular V gene segments have been previously shown to be localized in different anatomical sites. In this study, analysis of TCR-gamma/delta V gene segment usage performed on subjects from the time of birth through adulthood revealed striking age-related changes in the TCR-gamma/delta repertoire in peripheral blood. V delta 1+ gamma/delta T cells predominated in thymus as well as in peripheral blood at birth and then persisted as a relatively constant proportion of CD3+ PBL. However, V delta 2+ gamma/delta T cells that constitute a small proportion of the CD3+ cells in thymus and in peripheral blood at birth, then expand and account for the major population of gamma/delta T cells in PBL in adults. No parallel postnatal expansion of V delta 2+ cells in the thymus was observed, even when paired thymus-peripheral blood specimens were obtained on subjects between the ages of 3 d and 8 yr. The subset of V delta 2+ lymphocytes that was expanded in peripheral blood expressed high levels of CD45RO suggesting prior activation of these cells, consistent with the possibility that their expansion might have resulted from exposure to foreign antigens or superantigens. In contrast, V delta 1+ T cells in PBL showed no comparable increase in relative numbers and were either negative or expressed only low levels of CD45RO. Consistent with evidence for extrathymic peripheral expansion of selective TCR-gamma/delta subsets, no link between MHC haplotype and differences in the TCR-gamma/delta V gene usage between individuals was apparent, and identical twins displayed TCR-gamma/delta variable gene segment phenotypes that were strikingly different from one another. The elements that determine the TCR-gamma/delta repertoire in individuals are not known. It is possible that both thymic selection and extrathymic factors may influence the peripheral repertoire. Recently, TCR-gamma/delta+ lymphocytes have been shown to expand markedly in peripheral lymphoid tissues and infectious lesions in response to mycobacterial antigens, and a correlation between mycobacterial responses and TCR-gamma/delta V gene usage has been shown in mice. The data presented here demonstrated peripheral age-related changes in the gamma/delta repertoire and point to the importance of extrathymic expansion of specific gamma/delta subsets in generating the human TCR-gamma/delta repertoire.

Molecular recognition of lipid antigens by T cell receptors.

The T cell antigen receptor (TCR) mediates recognition of peptide antigens bound in the groove of major histocompatibility complex (MHC) molecules. This dual recognition is mediated by the complementarity-determining residue (CDR) loops of the alpha and beta chains of a single TCR which contact exposed residues of the peptide antigen and amino acids along the MHC alpha helices. The recent description of T cells that recognize hydrophobic microbial lipid antigens has challenged immunologists to explain, in molecular terms, the nature of this interaction. Structural studies on the murine CD1d1 molecule revealed an electrostatically neutral putative antigen-binding groove beneath the CD1 alpha helices. Here, we demonstrate that alpha/beta TCRs, when transferred into TCR-deficient recipient cells, confer specificity for both the foreign lipid antigen and CD1 isoform. Sequence analysis of a panel of CD1-restricted, lipid-specific TCRs reveals the incorporation of template-independent N nucleotides that encode diverse sequences and frequent charged basic residues at the V(D)J junctions. These sequences permit a model for recognition in which the TCR CDR3 loops containing charged residues project between the CD1 alpha helices, contacting the lipid antigen hydrophilic head moieties as well as adjacent CD1 residues in a manner that explains antigen specificity and CD1 restriction.

Self-recognition of CD1 by gamma/delta T cells: implications for innate immunity.

The specificity of immunoglobulins and alpha/beta T cell receptors (TCRs) provides a framework for the molecular basis of antigen recognition. Yet, evolution has preserved a separate lineage of gamma/delta antigen receptors that share characteristics of both immunoglobulins and alpha/beta TCRs but whose antigens remain poorly understood. We now show that T cells of the major tissue gamma/delta T cell subset recognize nonpolymorphic CD1c molecules. These T cells proliferated in response to CD1+ presenter cells, lysed CD1c+ targets, and released T helper type 1 (Th1) cytokines. The CD1c-reactive gamma/delta T cells were cytotoxic and used both perforin- and Fas-mediated cytotoxicity. Moreover, they produced granulysin, an important antimicrobial protein. Recognition of CD1c was TCR mediated, as recognition was transferred by transfection of the gamma/delta TCR. Importantly, all CD1c-reactive gamma/delta T cells express V delta 1 TCRs, the TCR expressed by most tissue gamma/delta T cells. Recognition by this tissue pool of gamma/delta T cells provides the human immune system with the capacity to respond rapidly to nonpolymorphic molecules on professional antigen presenting cells (APCs) in the absence of foreign antigens that may activate or eliminate the APCs. The presence of bactericidal granulysin suggests these cells may directly mediate host defense even before foreign antigen-specific T cells have differentiated.

An alpha-galactosylceramide C20:2 N-acyl variant enhances anti-inflammatory and regulatory T cell-independent responses that prevent type 1 diabetes.

Protection from type 1 diabetes (T1D), a T helper type 1 (Th1)-mediated disease, is achievable in non-obese diabetic (NOD) mice by treatment with alpha-galactosylceramide (alpha-GalCer) glycolipids that stimulate CD1d-restricted invariant natural killer T (iNK T) cells. While we have reported previously that the C20:2 N-acyl variant of alpha-GalCer elicits a Th2-biased cytokine response and protects NOD mice from T1D more effectively than a form of alpha-GalCer that induces mixed Th1 and Th2 responses, it remained to determine whether this protection is accompanied by heightened anti-inflammatory responses. We show that treatment of NOD mice with C20:2 diminished the activation of 'inflammatory' interleukin (IL)-12 producing CD11c(high)CD8+ myeloid dendritic cells (mDCs) and augmented the function of 'tolerogenic' DCs more effectively than treatment with the prototypical iNKT cell activator KRN7000 (alpha-GalCer C26:0) that induces Th1- and Th2-type responses. These findings correlate with a reduced capacity of C20:2 to sustain the early transactivation of T, B and NK cells. They may also explain our observation that C20:2 activated iNK T cells depend less than KRN7000 activated iNK T cells upon regulation by regulatory T cells for cytokine secretion and protection from T1D. The enhanced anti-inflammatory properties of C20:2 relative to KRN7000 suggest that C20:2 should be evaluated further as a drug to induce iNK T cell-mediated protection from T1D in humans.