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We analyzed the activity of the enhancer, the promoter and the silencer of the human CD4 gene during T cell development using transgenic mice. Immunofluorescence studies on thymic populations of mice carrying transgenes in various combinations of these regulatory DNA elements revealed that thymocytes control the CD4 gene in a different manner than mature peripheral T lymphocytes. The 5'-positive regulatory unit, consisting of the promoter and the 5' enhancer, is already active at the CD4-CD8-double-negative (DN) stage of development. However, its activity becomes lower in the double-positive and a fraction of the CD4+ CD8int/- cell population, indicating that an additional enhancer, located in either the first or the third intron of the CD4 gene, is required for CD4 gene expression in this population. The other studied regulatory element is the minimal CD4 silencer which inhibits CD4 gene expression in peripheral CD8 T lymphocytes. This silencer is inactive in the most immature DN thymocytes, which probably use a distinct silencer mechanism to down-regulate CD4 gene expression. Unexpectedly, the CD4 silencer is also active in CD4+ CD8int/- cells of the thymus, implying that an anti-silencer may be required to resume CD4 expression in this cell population. Altogether, the CD4 gene is regulated by several positive and negative regulatory mechanisms which come into play in a developmentally coordinated manner.

Five CD1 molecules are expressed in humans and it is unclear whether they have specialized or redundant functions. We found that sulfatide is a promiscuous CD1-binding ligand and have isolated T cell clones that are specific for sulfatide and restricted by distinct CD1 molecules. These clones have been used to compare the capacity of different CD1 to present the same glycolipid, to induce effector functions, and to form persistent immunogenic complexes. CD1a, CD1b, and CD1c molecules similarly load sulfatide on the cell surface without processing, and prime Th1 and Th2 responses. Stimulation by sulfatide-loaded CD1a persists much longer than that by CD1b and CD1c in living cells. Use of recombinant soluble CD1a confirmed the prolonged capacity to stimulate T cells. Moreover, other glycosphingolipids bind to all CD1, which suggests the presence of additional promiscuous ligands. Thus, group I CD1 molecules present an overlapping set of self-glycolipids, even though they are quite divergent from an evolutionary point of view.

T cells may recognize a large variety of ligands with different chemical structures. Recently, glycosphingolipids have also been shown to stimulate human T lymphocytes. Recognition of glycosphingolipids is restricted by the nonpolymorphic CD1 molecules, expressed by professional antigen-presenting cells and by macrophages infiltrating inflammatory sites. CD1 molecules have a structure resembling that of classical MHC class I molecules, with the terminal extracellular domains characterized by two antiparallel alpha helices placed on two hydrophobic pockets. The glycosphingolipids bound to CD1 insert the lipid tails in the two pockets and position the hydrophilic head on the external part of CD1. The TCR interacts with aminoacids present in the two alpha helices and with residues provided by the carbohydrate moiety of glycosphingolipids and discriminates their structural variations. T cells recognizing self-glycosphingolipids release proinflammatory cytokines and may have a pathogenetic role in autoimmune diseases such as multiple sclerosis.

In recent years, natural killer (NK) cells, as well as subpopulations of T cells, have been found to express diverse NK receptors (NKRs) for HLA class I molecules. We have characterized NKR phenotypes in lymphoproliferative disorders of NK or T-cell type. Peripheral blood of patients with lymphoproliferative disorders (n = 9) was analysed by multiparametric immunofluorescence flow cytometry with eight different antibodies against NKRs. Abnormal neoplastic cell populations from different types of NK or T-cell lymphoproliferative disorders lacked diversity in their NKR repertoires, i.e. all or none of the abnormal cells expressed individual NKRs and this expression occurred at single levels of intensity. This pattern of expression was specific for lymphoproliferative disorders as these resticted NKR repertoires were not found either in healthy donors (n = 9) or in patients with viral or autoimmune disease (n = 5). We conclude that NKRs are clonally expressed in lymphoproliferative disorders of NK or T-cell origin. NKR repertoires may represent a novel tool in diagnosing clonal disorders of NK and T-cell type.

The role of TNF and its type 1 receptor (TNFR1) in the pathogenesis of collagen-induced arthritis (CIA) was investigated in mice using two approaches. First, DBA/1 mice were treated after immunization with type II collagen by injecting TNFR1-IgG1 fusion protein to neutralize systemic TNF. CIA was prevented when treatment was administered shortly before the onset of clinical disease, suggesting that TNF is a crucial mediator in the late initiation phase of the arthritic process. In a second approach, TNFR1-deficient mice, generated by gene targeting and crossed to DBA/1, were used. These mice developed CIA with a low incidence and in a milder form. However, once a joint was afflicted, the disease progressed in this joint to the same end stage as that in wild-type mice. These data suggest that TNFR1 is the main transducer of TNF proinflammatory effects establishing CIA, but the progression of arthritis to tissue destruction and ankylosis is independent of TNFR1.

To study the relevance of gamma delta T cells in multiple sclerosis (MS) we analyzed the T cell receptor (TCR) gamma delta repertoire and the antigen reactivity of gamma delta clones isolated from cerebrospinal fluid (CSF). In T cell cultures derived from CSF we found an increased percentage of V delta 1+ cells as compared to peripheral blood of the same donors. Phenotypic analysis of cells from MS CSF with V gamma- and V delta-specific monoclonal antibodies (mAb) showed that the V delta 1 chain is most frequently associated with gamma chains belonging to the V gamma 1 family. Sequence analysis of TCR genes revealed heterogeneity of junctional regions in both delta and gamma genes indicating polyclonal expansion. gamma delta clones were established and some recognized glioblastoma, astrocytoma or monocytic cell lines. Stimulation with these targets induced serine esterase release and lymphokine expression characteristic of the TH0-like phenotype. Remarkably, these tumor-reactive gamma delta cells were not detected in the peripheral blood using PCR oligotyping, but were found in other CSF lines independently established from the same MS patient. Altogether, these results demonstrate that in the CSF there is a skewed TCR gamma delta repertoire and suggest that gamma delta cells reacting against brain-derived antigens might have been locally expanded.

Self lipid antigens induce selection and expansion of autoreactive T cells which have a role in immunoregulation and disease pathogenesis. Here we review the important biological rules which determine lipid immunogenicity. The impact of lipid structure, synthesis, traffic, membrane distribution and CD1 loading are discussed.

Self lipid antigens induce selection and expansion of autoreactive T cells which have a role in immunoregulation and disease pathogenesis. Here we review the important biological rules which determine lipid immunogenicity. The impact of lipid structure, synthesis, traffic, membrane distribution and CD1 loading are discussed.

Glatiramer acetate (GA) treatment for relapsing remitting multiple sclerosis (RRMS) leads to decreased GA-specific proliferative responses and a Th2 cytokine shift. To study a possible correlation between immunological and clinical responses to GA therapy, we prospectively followed RRMS patients clinically, by magnetic resonance imaging and by primary immunological assays. Fluctuation of GA-specific proliferative responses was significantly lower in treatment responders than in untreated patients, and GA-specific proliferative responses were increased during relapses. These associations suggest a possible causal relationship between immunological and clinical responses to GA therapy. Primary proliferation assays may thus be a useful marker for treatment response.

T cells may recognize a large variety of ligands with different chemical structures. Recently, glycosphingolipids have also been shown to stimulate human T lymphocytes. Recognition of glycosphingolipids is restricted by the nonpolymorphic CD1 molecules, expressed by professional antigen-presenting cells and by macrophages infiltrating inflammatory sites. CD1 molecules have a structure resembling that of classical MHC class I molecules, with the terminal extracellular domains characterized by two antiparallel alpha helices placed on two hydrophobic pockets. The glycosphingolipids bound to CD1 insert the lipid tails in the two pockets and position the hydrophilic head on the external part of CD1. The TCR interacts with aminoacids present in the two alpha helices and with residues provided by the carbohydrate moiety of glycosphingolipids and discriminates their structural variations. T cells recognizing self-glycosphingolipids release proinflammatory cytokines and may have a pathogenetic role in autoimmune diseases such as multiple sclerosis.

Lymphocytes recognize antigens with highly variable heterodimeric surface receptors. Although four distinct antigen receptors could in principle be produced by any lymphocyte, only one functional combination of receptor chains has thus far been found expressed on their surface. Examination of human gamma delta T cells revealed a population that violated this rule by expressing on their surface two distinct functional gamma delta T cell receptors (TCRs) that used different TCR gamma gene alleles. Thus, current models for T cell clonal selection may need modification, and a possible escape mechanism for autoreactive TCRs is suggested.

T cells recognize ligands of different chemical structures. Recently, it has become clear that also self glycosphingolipids and bacterial lipoglycans may act as T cell stimulatory ligands. This type of antigen recognition is restricted by the non-polymorphic CD1 molecules, which have a structure resembling that of classical MHC molecules. Glycolipids insert their hydrophobic lipid tails in two pockets below the antigen-binding groove and position their hydrophilic heads on the external part of CD1 molecules. TCR interacts with these carbohydrates and discriminates their structural variations. Glycolipid-specific T cells may provide protection during bacterial and parasite infection probably with different mechanisms: by secreting pro-inflammatory lymphokines, by the direct killing of infected target cells, and by helping specific B cells in Ig production. Lipoglycans represent excellent candidates for new anti-microbial vaccines due to their wide distribution in the microbial world and their structural composition which does not change and thus cannot give rise to escape mutants. Moreover, these vaccines might induce anti-microbial protective T cell responses in the whole population due to the non-polymorphic nature of CD1 presenting molecules.

In recent years, natural killer (NK) cells, as well as subpopulations of T cells, have been found to express diverse NK receptors (NKRs) for HLA class I molecules. We have characterized NKR phenotypes in lymphoproliferative disorders of NK or T-cell type. Peripheral blood of patients with lymphoproliferative disorders (n = 9) was analysed by multiparametric immunofluorescence flow cytometry with eight different antibodies against NKRs. Abnormal neoplastic cell populations from different types of NK or T-cell lymphoproliferative disorders lacked diversity in their NKR repertoires, i.e. all or none of the abnormal cells expressed individual NKRs and this expression occurred at single levels of intensity. This pattern of expression was specific for lymphoproliferative disorders as these resticted NKR repertoires were not found either in healthy donors (n = 9) or in patients with viral or autoimmune disease (n = 5). We conclude that NKRs are clonally expressed in lymphoproliferative disorders of NK or T-cell origin. NKR repertoires may represent a novel tool in diagnosing clonal disorders of NK and T-cell type.

To study the genes in the mouse background which predispose to the development of collagen-induced arthritis (CIA).T cell receptor beta transgenic (TCRbetaL) mice that have a T cell repertoire that predisposes to the development of CIA were used. Classic genetic studies and microsatellite gene mapping were done in (SWR-betaL x DBA/1)F2 hybrid mice.Besides TCRbeta, major histocompatibility complex class II, and Igh-C, at least 2 other genes are absolutely required for CIA development in these mice. A strict association of CIA with the presence of functional complement C5 allele (Hc1) was found, suggesting that Hc1 or a closely linked gene might be one of these essential genes.This study provides new evidence of the pathogenetic role of complement C5 in CIA. Furthermore, these transgenic mice may facilitate molecular identification of other genes that predispose to CIA.

To study the relevance of gamma delta T cells in multiple sclerosis (MS) we analyzed the T cell receptor (TCR) gamma delta repertoire and the antigen reactivity of gamma delta clones isolated from cerebrospinal fluid (CSF). In T cell cultures derived from CSF we found an increased percentage of V delta 1+ cells as compared to peripheral blood of the same donors. Phenotypic analysis of cells from MS CSF with V gamma- and V delta-specific monoclonal antibodies (mAb) showed that the V delta 1 chain is most frequently associated with gamma chains belonging to the V gamma 1 family. Sequence analysis of TCR genes revealed heterogeneity of junctional regions in both delta and gamma genes indicating polyclonal expansion. gamma delta clones were established and some recognized glioblastoma, astrocytoma or monocytic cell lines. Stimulation with these targets induced serine esterase release and lymphokine expression characteristic of the TH0-like phenotype. Remarkably, these tumor-reactive gamma delta cells were not detected in the peripheral blood using PCR oligotyping, but were found in other CSF lines independently established from the same MS patient. Altogether, these results demonstrate that in the CSF there is a skewed TCR gamma delta repertoire and suggest that gamma delta cells reacting against brain-derived antigens might have been locally expanded.

gamma delta T cells respond to a variety of microbial pathogens and transformed cells. Their limited receptor repertoire and activation by mycobacterial antigens resistant to proteases suggest that they may recognize nonpeptide antigens. We have tested a variety of nonpeptide molecules for stimulation of human gamma delta T cells. Synthetic alkyl phosphates, particularly monoethyl phosphate (MEP), selectively activated gamma delta T cells and stimulated their proliferation in vitro. All gamma delta T cells stimulated by MEP expressed V gamma 2/V delta 2 receptors. The purified natural ligand of mycobacteria is chemically similar to, though distinct from, MEP and contains a phosphate residue that is critical for biological activity. Recognition and expansion of a specific T-cell receptor-bearing population to non-peptide ligands is unprecedented among T cells. We suggest that MEP mimics small natural ligands capable of expanding one subset of gamma delta T cells and that this recognition of nonpeptide antigens may play an important role in human immunity to pathogens.

V gamma 9/V delta 2 cells represent a fraction of human gamma/delta cells that is expanded after birth in the periphery, carries markers of activated cells, and becomes a major population in peripheral blood. We found that these cells do not comprise a single population but actually represent two nested sets, the smaller of which, specific for Mycobacterium tuberculosis-pulsed antigen-presenting cells (APC), is contained in a larger set specific for an antigen found on the Molt-4 lymphoma. The larger set, representing 40-80% of all blood gamma/delta cells, is comprised of cells bearing the V gamma 9/C gamma 1 chain. Cells in the smaller, included set have an additional requirement for V delta 2 (and probably for certain permissive junctional regions, since a very small percentage of V gamma 9/V delta 2 cells do not react against mycobacteria-pulsed APC). Optimal stimulation by mycobacteria is dependent on the presence of APC, and is not restricted by classical major histocompatibility complex molecules. Some of the V gamma 9/V delta 2 mycobacteria-specific clones are also stimulated by APC pulsed with different bacteria, such as Listeria monocytogenes and Escherichia coli, indicating that the population includes several different patterns of reactivity. These data establish a relationship in humans between specificity and V gamma/V delta gene usage, and offer an explanation for the peripheral expansion of these gamma/delta cells.

In peripheral blood of normal donors a major fraction of γδ cells express a disulphide-linked (Cγ1) receptor with Vγ9 and Vδ2 gene products (Casorati 1989, Groh 1989). Since cells bearing Vγ9/Vδ2 TCR constitute only a minor fraction of γδ cells in the postnatal thymus (Casorati 1989) these cells might be selectively expanded in the periphery. The expansion of Vγ9/vδ2 cells may occur after stimulation with antigen (Parker 1990), or it may be dependent on encountering ligands that bind to the Vγ or vδ, irrespective of the specificities encoded by the V-(D)-J junctional sequences. To discriminate between these possibilities we investigated the antigen reactivity of Vγ9/vδ2 bearing cells. Here we describe two frequent reactivities which correlate with the V gene usage and might explain expansion in peripheral blood.

We have isolated T cell lines and clones from jejunal intestinal biopsies and peripheral blood of 7 coeliac disease (CD) patients, and 5 controls. T cells, expanded in the presence of PHA, rhIL-2 and feeder cells, were stained with the following mAbs: TR66 (anti-CD3), BMA032 (anti-αβ TCR), δ1 (anti-δ chain), δTCS1 (anti-Vδ1/Jδ1), BB3 (anti-Vδ2), TiτA (anti-Vτ9), C4-4All (anti-Vτ4), anti-CD4, and anti-CD8. The analysis of T cell lines showed that τ δ+ cells are increased in the gut of CD patients (range 15–52%) when compared to the controls (3–15%). Vδ1 was the most represented Vδ chain (47–76% of total τ δ cells), while Vδ2 was always below 21%. Vδ1 and Vδ2 negative τ δ cells were 11–42%. Vτ9 ranged from 18–68%, while Vτ4 varied between 14 and 41%. On the contrary, gut-τ δ cells derived from controls showed a higher variability in the Vδ and Vτ gene usage.

The ability of various cell types to secrete reactive nitrogen intermediates (RNI) upon functional activation was comparatively assessed. Neither in T lymphocyte clones mediating MHC class I or class II antigen-restricted killing via alpha/beta T cell receptor (TcR) or MHC-unrestricted killing via gamma/delta TcR, nor in peripheral blood mononuclear cells expressing natural killer or lymphokine-activated killer activity, target cell lysis was associated with detectable RNI production. Also, activated neutrophil granulocytes did not secrete RNI. In contrast, bone marrow-derived mononuclear phagocytes, activated to express tumoricidal activity, secreted marked RNI activity.

Killed Staphylococcus aureus strain Cowan I cells inhibit contact sensitivity to oxazolone in mice, when given intravenously 24–72 h before sensitization. With transfer experiments it was found that the cells responsible for the suppression are antigen-specific, nylon-adherent, resistant to antitheta serum + C, and sensitive to anti-mouse Ig serum + C. These suppressor B cells bear anti-oxazolone immunoglobulins and appear to exert their suppressive activity by preventing the contact sensitizer from reaching the specific reactive T cells.

This study was performed to investigate the cell-mediated immune response in burned patients with no septic episodes. The results show that burned patients with percentage body burn higher than 20 had an impaired lymphocyte reactivity to phytohaemagglutinin and conconavalin A. This hyporesponsiveness appeared on day 3–4 and in all cases reached its maximum on day 7–8 post burn, while recovery occurred between day 11 and 29 depending on the severity of the injury. The serum from immunodepressed patients was able to inhibit the response to phytohaemagglutinin and conconavalin A of normal lymphocytes. This immunosuppressive activity was present very early after injury (on day 1–2) and before the onset of lymphocyte hyporesponsiveness to mitogens and was no longer detectable on day 7–8 post burn, when patient lymphocytes showed the greatest hyporesponsiveness to mitogens. This late depression was due to T suppressor cells.

Polyclonal B cell activators (PBAs) such as purified protein derivative, lipopolysaccharide, Staphylococcus aureus strain Cowan I (StaCwI), and Pseudomonas aeruginosa inhibit contact sensitivity to oxazolone in mice when given 24 h before sensitization. This suppression, transferable from immunodepressed animals to oxazolone-sensitized recipients with immune serum, has been shown to be due to the early appearance of anti-hapten antibodies. These antibodies elicit T suppressor cells which release soluble factor(s) capable of inhibiting the passive transfer of contact sensitivity.

In vitro expanded T cell lines were used to determine whether antigen-specific cytolytic T lymphocytes are generated after infection with the intracellular bacterium, Listeria monocytogenes. Spleen cells from infected mice were cultured in the presence of syngeneic accessory cells, listerial antigen, and interleukin 2 containing supernatants. Cell lines were greater than 98% Thy-1+, L3T4-, Lyt-2+. Bone-marrow macrophages were used as target cells in two in vitro cytolytic assay systems. The Lyt-2+ T cells killed bone marrow macrophages only when infected with L. monocytogenes as assessed in a 4-hr 51Cr release assay and in an 18-hr neutral red uptake assay. Cytolysis was blocked by anti-LFA-1 and anti-Lyt-2 monoclonal antibodies. These cytolytic T cells produced interferon-gamma after co-stimulation with antigen, accessory cells, and recombinant interleukin 2. Bone marrow macrophages infected with Mycobacterium bovis were not killed by T cells from L. monocytogenes-infected mice but by T cell lines from M. bovis-infected mice, indicating that cytolysis was antigen specific. L. monocytogenes-infected target cells of different haplotype were lysed by the Lyt-2+ T cells. By using a low cell density split culture system, antigen-specific, H-2-restricted cytolytic T cells could be identified. These findings demonstrate that during infection with intracellular bacteria, Lyt-2+ T cells with cytolytic activity are generated that may be involved in antibacterial protection.

Cytokines have been suggested to act as intermediates between the immune and the central nervous system, but little is known about the type of cells synthesizing them in the brain. We have immortalized with oncogenic retroviruses primary brain cell cultures from mouse embryos and have generated clones of microglial cells that have been characterized. Three of the clones studied produce interleukin 1 (IL 1); IL 6 and tumor necrosis factor-alpha as assessed by biological assays and by Northern blot analysis. Our data raise the question on the role of these cytokines in the brain and suggest that early resident microglial cells might play an important role in development processes and in the adult brain.

Human thymocytes were sorted according to the expression of CD4 and CD8 molecules and clones representing the four subpopulations (DP, DN, and single CD4 or CD8 positive) were established. DP clones can be maintained for long periods in tissue culture and give rise to a variable percentage of SP variants. These variants, when isolated and further expanded, do not revert to a DP phenotype. DP clones express a functional TCR-CD3 complex, suggesting that this molecule can interact with the thymic microenvironment during T cell differentiation.

Mice were infected with Listeria monocytogenes and Lyt-2+ T cell clones capable of lysing Ag-primed bone marrow macrophages were established. In accordance with earlier findings obtained at the population level, some T cell clones were identified which lysed bone marrow macrophages of different MHC type provided the relevant Ag was present. This unusual target cell recognition was further analyzed using a T3+, L3T4-, Lyt-2+, F23+, KJ16+ T cell clone, designated L-28. Target cell lysis by this clone was Ag specific, apparently non-MHC restricted. In contrast, YAC cells and P815 cells were not lysed by clone L-28. However, lysis of irrelevant targets could be induced by anti-T3, F23, or KJ16 mAb. Furthermore, Ag-specific lysis was blocked by anti-Lyt-2 mAb and by F(ab)2 fragments of F23 mAb. In addition to its cytolytic activity, clone L-28 produced IFN-gamma after co-stimulation with accessory cells, Ag, and rIL-2 and conferred significant protection on recipient mice when given together with rIL-2. These data suggest that non-MHC-restricted Lyt-2+ killer cells generated during listeriosis are cytolytic T lymphocytes that interact with their target Ag via the T cell receptor/T3 complex and the Lyt-2 molecule and, furthermore, that these cells play a role in anti-listerial resistance. The possible relevance of IFN-gamma secretion and target cell lysis for antibacterial protection is discussed.

Purified protein derivative from Mycobacterium tuberculosis inhibits contact sensitivity to oxazolone in mice when given intravenously 24 to 72 h before the antigen. Transfer experiments showed that various types of suppressor cells occurred in the lymph nodes draining the site of sensitization: (i) anti-oxazolone idiotype + B lymphocytes, found at day 3 after sensitization, transferred suppression to syngeneic recipients at the moment of their sensitization; (ii) anti-idiotype B lymphocytes, found at day 3 after sensitization, transferred suppression to syngeneic recipients when sensitization of these animals had been performed 3 days before cell transfer; (iii) T lymphocytes, found only at day 6 after sensitization, inhibited the passive transfer of contact sensitivity, indicating that they were effective on the efferent phase of the immune response. These results indicate that purified protein derivative from M. tuberculosis interferes with contact sensitivity by activating a complex and multiple immunoregulatory circuit.

This study was undertaken in order to investigate the immunomodulatory effect of heat-killed Bacillus subtilis spores as well as of one soluble and one insoluble fraction obtained after their mechanical breakage. The results demonstrate that heat-killed spores, the soluble and the insoluble fraction enhance the primary PFC response to T-dependent antigens such as SRBC and HRBC, but not to T-independent antigens such as Ficoll-TNP. However, the same doses capable of enhancing the production of antibodies against T-dependent antigens were unable to modify the host CMI, evaluated in terms of contact sensitivity to oxazolone.

The effect of tuberculin purified protein derivative (PPD) on the development of plaque-forming cells (PFC) against bromelain-treated syngeneic mouse red blood cells (Br-MRBC) was studied in peritoneal cell cultures. The finding that PPD enhances the development of PFC to Br-MRBC, even under conditions where cell division is blocked by mitomycin C treatment, suggests that cell proliferation does not represent a necessary prerequisite for differentiation of precursor cells into autoantibody-forming cells.

We analyzed the V gamma and V delta gene usage in TCR-gamma/delta-bearing T cell clones isolated from human peripheral blood and postnatal thymus using V-specific mAbs and Southern and Northern analyses. In peripheral blood most of the gamma/delta cells express the V gamma 9-JP-C gamma 1 chain paired with a delta chain bearing the V delta 2 gene product. This heterodimer is very rare in the postnatal thymus, where a different and less restricted pairing of V gamma 9 and V delta 2 chains is found. These findings indicate that physical constraints cannot explain the overrepresentation of a particular V gamma 9-JP/V delta 2 heterodimer in the peripheral blood, and we discuss alternative mechanisms that may account for this differential distribution. In addition, this analysis allowed us to map the specificity of the delta TCS1 mAb to V delta 1-J delta 1 and to identify at least five different expressed V delta genes.

Mice were immunized intradermally with 10(7) irradiated Mycobacterium leprae organisms, and draining lymph nodes were collected after 4 weeks. Lymph node cells were restimulated in vitro with soluble M. leprae antigen and accessory cells. The resulting T-cell line was propagated in vitro in the presence of M. leprae antigen, accessory cells, and interleukin-2-containing supernatants from concanavalin A-stimulated rat spleen cells. Long-term cultured T cells were Thy-1+ L3T4- Lyt-2+ as revealed by analysis with the fluorescence-activated cell sorter. From this line, T-cell clones with the same phenotype were established. The T-cell clone A4 failed to secret interleukin-2 after stimulation with antigen and accessory cells, and its growth depended on exogeneous interleukin-2. A4 T cells produced gamma interferon in an antigen-specific, H-2-restricted, and interleukin-2-dependent way. Importantly, this T-cell clone was capable of lysing bone marrow macrophages presenting M. leprae antigen. Other T-cell clones as well as native Lyt-2+ T cells from M. leprae-immunized mice were also capable of lysing bone marrow macrophages expressing M. leprae antigens. These findings suggest that specific Lyt-2+ T cells participate in the immune response to M. leprae. It is postulated that cytolysis of M. leprae-infected macrophages or Schwann cells contributes to protection against and pathogenesis of leprosy.

✓ The use of tumor-infiltrating lymphocytes in the treatment of central nervous system (CNS) neoplasms has met with serious obstacles due to difficulty of culture and poor characterization. Since in other tumors the therapeutic effects of tumor-infiltrating lymphocytes have been shown to rely on T-cell receptor engagement, the authors addressed the question as to whether expression of T-cell receptor variable (V) domains in cultured tumor-infiltrating lymphocytes from CNS is different from that of autologous cultured peripheral blood mononuclear cells. Infiltrating lymphocytes from CNS neoplasms, including primary malignancies, metastatic cancers, and meningiomas, were cultured in the presence of interleukin-2 and anti-CD3 monoclonal antibodies (MoAb's) in order to obtain optimum growth of T cells. Autologous peripheral blood mononuclear cells from the same patients were similarly cultured. After 4 to 5 weeks of culture, 97.3% ± 2.6% (mean ± standard deviation) of the resulting cell populations were CD3-positive lymphocytes. The expression of T-cell receptor V domains was then studied by using a panel of 12 MoAb recognizing gene products from T-cell receptor V-α 2, V-β 5, 6, 8, and 12, V-γ 4 and 9 families, and from two subfamilies of V-δ 2. Remarkably, in over 70% of all paired measurements, percentages of T cells expressing discrete T-cell receptor V-gene products were found to be virtually identical in tumor- and peripheral blood-derived cultured cell populations, with differences never exceeding 1%. In contrast, a different expression of individual V-gene products, concerning both α/β and γ/δ T-cell receptors, could be detected between cultured tumor-infiltrating lymphocytes and autologous peripheral blood-derived T lymphocytes in seven of 12 patients. In two cases, significant differences between the two populations were also observed in the proliferative responses obtained upon stimulation with staphylococcal enterotoxins that trigger defined V-β T-cell receptors. Altogether, these data suggest that the T-cell receptor repertoire of cultured tumor-infiltrating lymphocytes from CNS tumors, suitable for use in adoptive immunotherapies, differs from that of autologous cultured peripheral blood mononuclear cells.

Lyt-2+ T cell clones were established from Listeria monocytogenes-infected mice. The clones secreted IFN-gamma after stimulation with spleen cells from L. monocytogenes-infected mice plus IL-2. Spleen cells from normal mice were not stimulatory. Furthermore, cloned T cells lysed L. monocytogenes-infected macrophages. Cytolysis was antigen-specific and H-2K-restricted. These findings suggest a role for specific cytotoxic T cells in the immune response to intracellular bacteria.

A concise synthesis of alpha-sulfatide 1, an analogue of natural glycolipid antigens with potential anti-tumor activity, was performed. Two different approaches to the alpha-glycosidic bond were explored, resulting in a high yield and excellent stereoselectivity. Compound 1 combines the structural features of sulfated beta-GalCer (sulfatide) and alpha-GalCer, which activate specific T cells. alpha-Sulfatide 1 was stimulatory for CD1d-restricted semi-invariant Natural Killer T (iNKT) cell clones, although less potent than alpha-Gal-Cer, while it was not recognized by CD1a-restricted sulfatide-specific T cells.