Transient monocyte release of interleukin-10 in response to traumatic brain injury.

A significant component of the immune response to trauma results in the systemic presence of cytokines which have the potential to suppress the patient's immune response to infection and contribute to post-injury complications. We assayed peripheral blood leukocytes obtained from 10 patients with head trauma to determine their production of interleukin (IL). Serum was assayed for the presence of IL-10, TGFbeta1, and IFNgamma by ELISA. Peripheral blood leukocytes were screened for intracellular IL-10 and IFNgamma by fluorescence-activated flow cytometry, and cytokine-specific mRNA was detected by the polymerase chain reaction. We detected an immediate, but transient, presence of IL-10 in the sera of all 10 patients who suffered head trauma. IL-10-specific intracytoplasmic immunofluorescence was also detected immediately after injury in peripheral blood monocytes, but not in lymphocytes or granulocytes. IL-10-specific mRNA was detected in peripheral blood leukocytes in only 50% of patients immediately after injury, when the highest serum levels of IL-10 were observed. Our data indicates that release of pre-formed IL-10 by monocytes contributes to the presence of IL-10 found in patient peripheral blood immediately after head injury.

Delineation by use of specific monoclonal antibodies of the T-cell receptor and major histocompatibility complex interaction sites on the superantigen toxic shock syndrome toxin 1.

Murine monoclonal antibodies (MAbs) specific for toxic shock syndrome toxin 1 (TSST-1), a bacterial superantigen, showed the ability either to detect TSST-1 bound to histocompatibility locus antigen (HLA)-DR molecules or to inhibit TSST-1 binding to HLA-DR. A MAb capable of detecting DR-bound TSST-1 could also inhibit the toxin-induced activation of a T-cell receptor Vbeta15-expressing murine T-cell hybridoma. Alternatively, MAbs with specificity for the HLA-DR association site could present TSST-1 in vitro, stimulating CD4+ human T cells to proliferate. These functional activities correlated directly with with MAb specificity for HLA-DR versus T-cell receptor Vbeta interaction sites on TSST-1 as determined by reactivity with a panel of recombinant TSST-1 mutant molecules. Therefore, these MAbs discriminate the superantigen functional sites on the TSST-1 molecule and constitute reagents with the property of being potent modulators of the toxic activity of TSST-1.

Identification of class II major histocompatibility complex and T cell receptor binding sites in the superantigen toxic shock syndrome toxin 1.

Superantigens, in association with class II major histocompatibility complex (MHC) molecules, activate T cells bearing particular beta chain variable domains of the T cell receptor (TCR). Unlike conventional peptide antigens, superantigens bind as intact proteins to TCR and MHC molecules outside their peptide binding sites. To characterize these interactions at the molecular level, random point mutations were generated in the gene encoding toxic shock syndrome toxin 1, a bacterial superantigen associated with toxic shock syndrome. Functionally impaired mutants were identified based on their lack of murine and human T cell stimulatory activities, and experiments analyzing binding to human histocompatibility leukocyte antigen-DR molecules differentiated residues involved in MHC from TCR binding. The results showed that the great majority of mutations are clustered in two distinct regions of the toxic shock syndrome toxin 1 molecule. The class II MHC binding site is located in the hydrophobic region of the NH2-terminal domain, and the TCR binding site is primarily in the major central groove of the COOH-terminal domain. These studies provide insight into the interactions necessary for superantigen-mediated disease in humans.

Selection for T-cell receptor V beta-D beta-J beta gene rearrangements with specificity for a myelin basic protein peptide in brain lesions of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system in which a restricted cellular immune response has been observed. In order to establish whether such T cell responses are likely to be antigen-specific particularly with regard to myelin basic protein (MBP), we analysed T-cell receptor (TCR) gene rearrangements directly from MS brain plaques, using the polymerase chain reaction on reverse transcribed messenger RNA, and compared these with TCR of previously described MBP-specific T cell clones from MS and the rat model experimental allergic encephalomyelitis. Rearranged V beta 5.2 genes were detected in the brains of all patients who were HLA DRB1*1501, DQA1*0102, DQB1*0602, DPB1*0401. The V beta 5.2-D beta-J beta sequences in these MS brain plaques revealed five motifs. One of the common motifs was identical to that described for the VDJ region of a V beta 5.2 T-cell clone. This clone was from an MS patient who was HLA DRB1*1501, DQB1*0602, DPB1*0401, and it was cytotoxic towards targets containing the MBP peptide 89-106 (ref. 1). The deduced amino-acid sequence of this VDJ rearrangement, Leu-Arg-Gly, has also been described in rat T cells cloned from experimental allergic encephalomyelitis lesions, which are specific for MBP peptide 87-99 (ref. 2). VDJ sequences with specificity for this MBP epitope constitute a large fraction (40%) of the TCR V beta 5.2 N(D)N rearrangements in MS lesions. The capacity of rat T cells with these VDJ sequences to cause experimental allergic encephalomyelitis and the prevalence of such sequences in demyelinated human lesions indicate that T cells with this rearranged TCR may be critical in MS.

Clonal expansions of activated gamma/delta T cells in recent-onset multiple sclerosis.

Multiple sclerosis (MS) is a chronic disease characterized by focal demyelination of the white matter of the brain and spinal cord. Central nervous system damage appears to be mediated by infiltrating T lymphocytes and macrophages, and a central role for autoreactive CD4+ T cells has been proposed. However, the initial immune events that lead to the chronic process of MS remain unidentified. We now present evidence that a subset of T lymphocytes bearing gamma/delta T-cell antigen receptors has been activated in patients with recent-onset disease. Cells recovered from the cerebrospinal fluid of subjects with MS were cultured for short periods of time in medium supplemented with T-cell growth factors. Expansions of V delta 1 and V delta 2 T-cell receptor-bearing lymphocytes were found only in cell populations obtained from subjects with recent-onset disease. Similar populations were not expanded in subjects with chronic MS or other neurological diseases. Junctional region sequencing showed the expanded gamma/delta T cells to be oligoclonal in nature, suggestive of specific stimulation by antigen. These results reveal a fundamental difference in the immunopathogenesis of acute vs. chronic disease and provide additional insight into the autoimmune nature of MS.

Thymocyte subpopulations during early fetal development in the BALB/c mouse.

Phenotypic analysis of thymocytes during murine fetal development may be of use in determining the pathways of thymocyte differentiation. The expression of the functionally significant molecules Lyt-2 (CD8), L3T4 (CD4), and the TCR has already been described. However, mAb specific for several other murine lymphocyte surface markers are now available and, although these have been used to characterize adult thymocytes, a detailed analysis of fetal thymocytes with these antibodies has not previously been undertaken. In this study, we have used mAb specific for Thy-1, J11d, Pgp-1, and the IL-2R, in addition to those for Lyt-2 and L3T4, to identify subpopulations of early fetal thymocytes. By using two-color flow cytometric analysis of cells obtained from fetal thymuses on sequential days of gestation, we have been able to follow the development of various subpopulations through early fetal ontogeny. Our data indicate that the earlier thymocytes are found in the J11d+/Pgp-1+ subset which is abundant at fetal day 14 but constitute a numerical minority by day 16.

Split tolerance induced by the intrathymic adoptive transfer of thymocyte stem cells.

The intrathymic transfer of semiallogeneic CD4/CD8 double-negative (DN) thymocyte stem cells into irradiated host mice resulted in a transient state of chimerism in adoptive host thymus, spleen, and lymph nodes. Host-derived T cells, isolated from the thymus and periphery of the chimeric mice, were found to be specifically nonresponsive to the MHC antigens of the semiallogeneic DN donor in cytotoxicity assays. This nonresponsiveness was not permanent, but persisted as long as appreciable numbers of Thy-1 alloantigen-positive progeny of the DN donor cells could be detected in the spleen and lymph nodes of adoptive host mice. FACS sorting of DN donor cells before intrathymic transfer indicated that nonresponsiveness could be induced by Thy-1+ cells and was therefore not attributable to contaminating thymic macrophages, dendritic cells, or B cells. When FACS-sorted Thy-1+ (bm5 x bm12)F1 DN cells were transferred intrathymically into C57BL/6 hosts, nonresponsiveness to DN donor MHC class I but not class II alloantigen (split tolerance) was observed. These experiments were repeated using FACS-sorted Thy-1+ DN donor cells that were semiallogeneic to the irradiated adoptive host at either MHC class I or class II locus with similar results. Limiting dilution analysis showed that host-derived CTL precursors were tolerant of DN donor MHC class I alloantigen and no evidence for the involvement of suppressor T cells was found. The data indicate that murine thymocytes themselves are capable of tolerizing to MHC class I but not class II alloantigen after intrathymic transfer. The implications for intrathymic T cell differentiation and maintenance of self tolerance are discussed.

Expression of T cell antigen receptor beta-chains on subsets of mouse thymocytes. Analysis by three-color flow cytometry.

The expression on adult mouse thymocytes of a T cell antigen receptor beta-chain epitope, recognized by the antibody F23.1, has been studied by three-color flow cytometry. Low density F23.1 staining was found mainly on CD4+8+ thymocytes. High density staining was mainly on CD4+8- and CD4-8+ cells. Variable proportions of CD4-8- cells were also F23.1+. Among CD4-8+ cells, F23.1 was expressed only on the J11d- subset with mature T cell function. We conclude that many subpopulations of thymocytes express antigen receptors and are candidates for the population subject to thymic selection, but at present no single subpopulation makes a convincing claim.

Transient expression of IL-2 receptor precedes the differentiation of immature thymocytes.

The growth of mature T lymphocytes after activation by antigen is regulated by the binding and endocytosis of interleukin-2 (IL-2). In the thymus, approximately 50% of adult thymocytes that carry neither the CD4 nor the CD8 antigen and day 14-15 fetal CD4-8- thymocytes express receptors for IL-2(IL-2R). The CD4-8- (double-negative) subpopulation of thymocytes contains the precursors of cells that can differentiate along an unknown pathway into thymocytes bearing either CD8 or CD4, with the characteristics of mature T lymphocytes. The basis for IL-2R expression by double-negative thymocytes is unclear as they appear to lack a functional T-cell receptor/CD3 complex through which activation of peripheral T cells is mediated. The argument for a role for IL-2 in thymocyte differentiation has also been complicated by conflicting reports on the inability or capability of double-negative thymocytes to respond to IL-2 in vitro. At present, both the nature of the stimuli within the thymic micro-environment which induce IL-2R expression and its relevance to thymocyte differentiation are not known. We show here that the IL-2R-bearing subset has a greater potential to differentiate into phenotypically mature T lymphocytes than do IL-2R-negative thymocytes. In addition, progeny of IL-2R-negative donor cells transiently express IL-2R in the thymuses of adoptive hosts before generating CD8 and/or CD4-positive thymocytes. These results identify the IL-2R-positive cells as a more differentiated double-negative thymocyte subset on the pathway to mature T lymphocytes.

Differentiation potential of subsets of CD4-8- thymocytes.

Precursor T cells in the thymus are contained within a subpopulation of thymocytes that lack the markers CD4 and CD8. We have examined the heterogeneity of these cells by flow cytometric analysis, and defined four subpopulations using the cell surface markers Thy-1, J11d and the IL-2 receptor (IL-2R). The J11d+ subset of CD4-8- cells all bear the antigen Thy-1, and some express the IL-2R. Staining and RNA analysis of J11d+ cells suggest that some express receptors of the CD3 gamma delta type, but none express CD3 alpha beta receptors. In fetal thymus organ culture, the J11d+ cells diversify to form 'cortical type' CD4+8+ cells and 'medullary type' cells expressing either CD4 or CD8; in vivo they repopulate the thymus of an irradiated host and seed the periphery with T cells. In contrast, the J11d- subset of CD4-8- thymocytes do not all bear Thy-1 and none express the IL-2R, but some express antigen receptors of the CD3 alpha beta type. They have more limited diversification potential in organ culture, and in vivo fail to recolonize the irradiated host in a homing-independent assay. We conclude that they are not precursor T cells, but rather a side-branch from the main line of T cell differentiation.

Different structural constraints for recognition of mouse H-2Kd and -Kk antigens by alloimmune cytolytic T lymphocytes.

We have constructed a new series of hybrid genes among the H-2Kd,-Kk, and -Kb. The site of recombination occurs in the third exon, encoding the alpha 2 domain, and divides this domain into two parts, alpha 2A and alpha 2B. The novel genes differ only in the COOH-terminal half of the alpha 2 domain, i.e., the alpha 2B region. This region, comprising residues 142-182, contains a limited number of amino acid differences between the three alleles. The hybrid genes have been introduced into 1T 22-6 cells (H-2q), and cell surface expression of hybrid antigens was verified. Cells expressing different types of hybrid antigens have been examined for their susceptibility to lysis by cytotoxic T lymphocytes directed either against the H-2Kd antigen or the H-2Kk antigen. Our results show that the alpha 1 and alpha 2A domains of the H-2Kk antigen can constitute target molecules for alloimmune anti-Kk T cells, whereas the alpha 2B region, when exchanged for Kd or Kb sequences, plays only a limited role. In contrast, the alpha 1 and alpha 2A domains of Kd are not sufficient to be recognized by alloimmune anti-Kd T cells. In this instance, the alpha 2B domain seems to play an essential role. This region has undergone several amino acid substitutions involving charged residues.

Clonal analysis of cytolytic T lymphocyte-mediated lysis of target cells with inducible antigen expression: correlation between antigen density and requirement for Lyt-2/3 function.

The lysis by allospecific cytolytic T lymphocytes (CTL) of the BALB/c lymphoma ST-4.5, a cell line that can be induced by interferon-gamma (IFN-gamma) to express increased amounts of major histocompatibility complex (MHC) class I antigens, was investigated. Culture of ST-4.5 in IFN-gamma increased the surface expression of Kd molecules from originally low levels and Dd from undetectable amounts by approximately fivefold as determined by fluorescence-activated cell sorter (FACS) analysis, whereas the levels of several other antigens (Ld, I-Ad, Thy-1, Lyt-2, L3T4, and LFA-1) were not affected. The lysis of ST-4.5 by Dd- and Ld-specific CTL clones correlated with the expression of those antigens on target cells as determined by both FACS and biochemical analysis. Lysis of ST-4.5 by CTL clones specific for Kd antigen fell into two distinct groups: those that could lyse targets cultured either normally or in IFN-gamma, and those that could only lyse targets that had been precultured in IFN-gamma. The apparent sensitivity to antigen exhibited by the Kd-specific CTL clones predicted their sensitivity to inhibition of target lysis by anti-Lyt-2/3 antibody. Those CTL clones that were only active against ST-4.5 expressing higher amounts of surface antigen (resulting from IFN-gamma preculture) were readily inhibited by anti-Lyt-2/3 antibody, whereas those CTL capable of lysing normally cultured targets having lower amounts of surface antigen were heterogeneous in their sensitivity to anti-Lyt-2/3; some were inhibitable, whereas others were resistant. In addition, another CTL clone that was resistant to inhibition by anti-Lyt-2/3 alone was readily inhibited by a synergistic combination of anti-Lyt-2/3 plus anti-Kd (but not anti-Dd or Ld) antibodies. These results indicate that CTL antigen receptor sensitivity to (or affinity for) antigen and the level of specific antigen expression by the target cell may both be important criteria in assessing Lyt-2/3 molecule function in CTL-mediated cytolysis. The function of recognition-associated molecules such as Lyt-2/3 may be to strengthen and increase the number of receptor-ligand binding events that facilitate CTL-target membrane interactions that lead to the lysis of the target cell.

Effect of prostaglandin E2 on the gamma-interferon induction of antigen-presenting ability in P388D1 cells and on IL-2 production by T-cell hybridomas.

The effect of prostaglandin E2 on the gamma-interferon (IFN-gamma)-mediated induction of Ia expression and antigen-presenting activity in macrophage cell lines was studied. Using a lymphokine preparation obtained from the T-cell hybridoma FS7-20.6.18 (known to produce interferon) to induce the expression of Ia in P388D1 cells, the influence of PGE2 on this phenomenon was studied. Screening of the cell cultures by indirect immunofluorescence using an anti-I-Ad monoclonal antibody confirmed the inhibitory effect of PGE2 in the induction of I-Ad. However, the inhibition of the antigen-presenting ability of these cells, as measured by their capacity to stimulate interleukin 2 (IL-2) production by antigen-specific, I-region-restricted (Ag/I) T-cell hybridomas, was more difficult to demonstrate and was only evident when using low concentrations of Ia-inducing lymphokines or when using "low avidity" T-cell hybridomas. The latter were distinguished by the limited response (in the form of IL-2 production) that was observed when they were tested with P388D1 cells that had been induced with IFN-gamma. By contrast, PGE2 had profound inhibitory effects on the ability of T-cell hybridomas to secrete IL-2 in response to Ag/I or concanavalin A. These results suggest that although PGE2 inhibits the full induction of Ia on macrophages, it has little effect on the induction of Ag/I presentation by the same cells, probably because most T cells require relatively low levels of Ia on the surface of presenting cells. T-cell responses to Ag/I are inhibited, however, because of the effects of PGE2 on the T cells themselves.

Antigen recognition by H-2-restricted T cells. II. A tryptic ovalbumin peptide that substitutes for processed antigen.

A 17-amino acid tryptic peptide of chicken ovalbumin, designated P323-339, that substituted for processed antigen when presented by glutaraldehyde prefixed accessory cells to specific I-restricted T hybridomas was characterized. The peptide antigen could not be demonstrated to have any specific or stable interactions with accessory cell Ia antigens by either direct binding or functional assays for inhibition of specific T cell activation. In addition, the T cell receptor for I-restricted antigen had no affinity for free antigen alone. A rabbit antibody specific for the antigenic peptide inhibited presentation when introduced before but not after binding of the peptide to accessory cells. These results extend our earlier finding that accessory cell-mediated processing of chicken ovalbumin can be completely explained by the fragmentation of the native molecule into smaller m.w. peptides, and suggests that if an antigen/Ia complex is important in T cell activation, it forms significantly only in the presence of the T cell receptor for I-restricted antigen.