Indirect costs of research--beyond self-interest.
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Costs and Cost Analysis↗
Biomedical subjects
F W Fitch
Publications and source records attributed to F W Fitch.
The gamma chain of the high-affinity receptor for IgE is a major functional subunit of the T-cell antigen receptor complex in gamma delta T lymphocytes.
T-cell activation is a consequence of the clonotypic T-cell antigen receptor (TCR) binding to an antigen followed by signal transduction via the invariant subunits of the TCR/CD3 complex. gamma delta TCR cells are a small subset of T cells that populate both the epithelial and lymphoid tissues and have unique antigen specificity and function. However, the composition of invariant chains within the gamma delta TCR/CD3 complex has not been well characterized. Here we report that, unlike the majority of alpha beta T cell, gamma delta T cells isolated from spleen and intestinal epithelial tissue express high levels of the gamma chain of the high-affinity receptor for IgE (Fc epsilon RI gamma) as one invariant subunit of their TCR/CD3 complex. Fc epsilon RI gamma exists as both a homodimer and a heterodimer associated with the TCR zeta chain. Moreover, stimulation of the gamma delta TCR results in rapid tyrosine phosphorylation of Fc epsilon RI gamma. Our results suggest that utilization of distinct receptor signaling components may enable the coupling of antigen stimulation to the activation of different signal transduction pathways in alpha beta and gamma delta T cells.
Animals↗
Cytolytic activity of murine IL-2-producing CD4+ and CD8+ T cell clones cycles in response to IL-2.
Alloreactive or OVA-reactive cloned murine CD4+ or CD8+ T cells that produce IL-2 exhibit greatly reduced cytolytic activity after being cultured with high concentrations of rIL-2. Furthermore, such cells fail to produce lymphokines or proliferate when stimulated with Ag. The duration of this unresponsiveness to Ag correlates with the concentration of rIL-2 to which the cells were exposed; higher concentrations of rIL-2 prolong the period of unresponsiveness. The presence of ionomycin during the cytolytic assay restores lytic activity to cells rendered unresponsive by exposure to rIL-2. These results suggest that rIL-2-induced unresponsiveness to Ag is a consequence of impairment of a calcium-dependent signal important for cytolysis, proliferation, and lymphokine production. Thus, IL-2 appears to be an important lymphokine that regulates T cell responses downward as well as upward.
Animals↗
Accessory molecules involved in antigen-mediated cytolysis and lymphokine production by cytotoxic T lymphocyte subsets. I. Identification of functions for the T cell surface molecules Ly-6C and Thy-1.
The murine T cell surface molecules Ly-6C and Thy-1 are genetically and structurally distinct, yet they share two interesting properties: both are attached to the plasma membrane through a glycophosphatidylinositol linkage, and some mAb reactive with these molecules can activate T cells. Although mAb for Ly-6C and Thy-1 appear to mimic the function of physiologic ligands, direct evidence for the existence of these putative ligands has not been presented. In this report, we describe CTL clones that use Ly-6C and Thy-1 as accessory molecules for activation of cytolysis and the production of IFN-gamma based on inhibition of these functions with mAb. These studies were facilitated by the derivation of a nonactivating hamster IgM mAb specific for Ly-6C. CTL clones that use Ly-6C and Thy-1 as accessory molecules include a subpopulation of the previously described CD8+ alloreactive CTL that are not inhibited by mAb reactive with CD8, a CD8+ TCR-alpha/beta+ T cell clone specific for HSV glycoprotein D, and a CD4-CD8- TCR-gamma/delta+ T cell clone specific for HSV glycoprotein I. The role of Ly-6C and Thy-1 in target cell recognition is to some degree tissue-specific with respect to the APC/target cell. A mAb specific for Ly-6C appears to inhibit activation by prevention of adhesion between the effector cells and the target cells. This is the most direct evidence to date of a functional ligand for Ly-6C.
Animals↗
The good old days--past, present, and future.
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Allergy and Immunology↗
Cytolytic activity of murine CD4+ T cell clones correlates with IFN-gamma production in mouse strains having a BALB/c background.
CD4+ murine T cell clones were derived from various strains of mice, and their pattern of lymphokine secretion and cytolytic activity was compared. Limiting dilution cultures were established with lymph node cells from mice sensitized with OVA. Alloreactive CD4+ T cell clones also were derived in limiting dilution cultures prepared with naive BALB/c-H-2dm2 lymph node cells stimulated with irradiated BALB/c splenocytes. A total of 24 days elapsed between establishment of cultures and analysis of lymphokine production and cytolytic activity. Cytolytic capacity was assessed by using target cells that had been pulsed with Ag or coated with anti-CD3 mAb. We observed that: 1) the frequency of OVA-reactive T cells from various mouse strains was approximately the same; 2) both Th1 and Th2 cells as well as cells not encompassed within these categories could be lytic if derived from DBA/2, B10.D2, B10.A, C57BL/10, or C57BL/6 mice; and 3) the vast majority of CD4+ cloned T cells derived from BALB/c, BALB/c-H-2dm2, BALB.B, or BALB.K that did not produce IFN-gamma (including Th2 cells) did not exhibit cytolytic activity, whereas most clones derived from these strains that produced IFN-gamma were cytolytic. These observations indicate that both Th1 and Th2 cells from several mouse strains express cytolytic activity. Such cytolytic activity was not restricted to clones maintained in long term cultures. However, genes outside the MHC appeared to regulate the cytolytic activity of T cells. In particular, CD4+ T cell clones which did not produce IFN-gamma were not cytolytic when they were derived from BALB/c mice and mutant or MHC congenic inbred mice having a BALB background. Cytolytic activity of CD4+ T cells, in addition to the pattern of lymphokine production, may be important in graft rejection and in immune responses to infectious diseases.
Animals↗
Multiple components of the T cell antigen receptor complex become tyrosine-phosphorylated upon activation.
Triggering of the multicomponent T cell antigen receptor (TCR) complex results in several biochemical processes which are critical for the functional activation of T lymphocytes. One common process is the tyrosine phosphorylation of several proteins, including the TCR zeta chain. Here we show that in addition to TCR zeta, other subunits (CD3 gamma, CD3 delta, and CD3 epsilon) of the TCR complex can also be tyrosine-phosphorylated in response to antigen receptor stimulation. This rapid phosphorylation was detected in several mature murine T cell subsets, including CD4+ type 1 and 2 helper cells (TH1 and TH2). Therefore, tyrosine phosphorylation of multiple TCR components in addition to TCR zeta may be an important event during the initiation of the signaling cascade leading to T cell activation.
Animals↗
Anergized T cell clones retain their cytolytic ability.
CD4+ T cells have been described to have both helper and lytic function. The helper function of Th1 cells in particular can be inactivated by inducing the T cell into a state of nonresponsiveness in which the T cell is no longer capable of producing IL-2 or proliferating in an autocrine way to a conventional antigenic stimulus. To determine whether the lytic ability of Th1 cells can also be rendered nonfunctional upon anergy induction, we induced Th1 clones into a nonresponsive state and tested their ability to lyse target cells in an Ag-specific and MHC class II-restricted manner. We show that cells newly induced into an anergic state were able to lyse target cells nonspecifically. This effect was short-lived and after resting in culture media, the cells regained their ability to lyse target cells in an Ag/MHC-specific manner, and this ability was comparable to normal resting T cells. In contrast, the helper function of these cells remained nonresponsive, and the cells were unable to proliferate or to secrete IL-2 in response to the same antigenic stimulus used for lysis. Therefore, the lytic pathway appears to be regulated separately from the proliferative/lymphokine pathway(s) and is not affected long-term by an anergic stimulus.
Animals↗
Identification and propagation of a putative immunosuppressive orphan parvovirus in cloned T cells.
A putative parvovirus related to minute virus of mice (MVM), but distinct from MVM-prototype and MVM-immunosuppressive, was identified, using serologic techniques and Southern blot analysis, in maintenance cultures of established T cell clones. This putative viral agent resulted in a lytic infection of cloned L3 cytotoxic T cells but was unable to produce a productive infection in BHK.21 or EL-4(G) cells. Moreover, maintenance cultures of several distinct subsets of cloned T cells apparently contaminated with this putative viral agent contained poorly growing cells and erythrocyte aggregates. The aggregation of mouse erythrocytes appeared to be a reliable indicator of infection with this putative virus and may be related to the ability of this agent to agglutinate mouse erythrocytes. This putative virus also was found to inhibit the proliferative response of certain cloned T cells to IL-2 and Ag. Viremic mice and secondary MLC supernatant were identified as two potential sources of contamination and represent ways of propagating this agent in vitro. The finding that this agent interferes with the ability of T cell clones to thrive and, therefore has the potential to alter immune responses, emphasizes the importance of identifying and excluding parvoviral infections in cultures of murine T lymphocytes.
Animals↗
Differential regulation of murine T lymphocyte subsets.
Signaling pathways in T lymphocytes have been incompletely characterized. It is evident that differences exist among the T cell subsets. We have defined several distinct mechanisms that affect differentially the activities of murine T lymphocyte clones representing various CD4+ and CD8+ subsets: Interferon-gamma (IFN-gamma) inhibits proliferation of but not lymphokine production by TH2 cells. IL-10 inhibits antigen-presenting cell (APC)-induced lymphokine production by TH1 cells but not by TH2 cells. Murine TH1 and TH2 clones proliferate optimally in response to distinct APC populations. TH1 and TH2 clones utilize different TCR-associated signaling pathways. High concentrations of antigen (or anti-TCR mAb) inhibit IL-2-induced proliferation (but not lymphokine production) by TH1 and cytolytic T lymphocyte (CTL) clones only. Exposure of TH1 clones (but not TH2 clones or CD8+ CTL clones) to IL-2 induces unresponsiveness to antigen. TH1 and TH2 clones as well as CD8+ clones can be cytolytic, but not all T cells use the same cytolytic mechanisms. CD4+ clones from some mouse strains are not cytolytic if they do not secrete IFN-gamma. Understanding the mechanisms that differentially regulate the various kinds of T cells, in addition to providing insights into the molecular events associated with activation of those subsets, should facilitate modulation of their activities in vivo, making it possible to influence favorably the outcome of disease processes.
Animals↗
A murine CD4-, CD8- T cell receptor-gamma delta T lymphocyte clone specific for herpes simplex virus glycoprotein I.
The role of TCR-gamma delta T lymphocytes in immune responses is currently not well understood. TCR-gamma delta cells have a limited repertoire suggesting that TCR-gamma delta T a limited number of evolutionarily conserved Ag such as nonpolymorphic MHC and heat shock proteins. TCR-gamma delta T lymphocytes appear in enhanced numbers in skin lesions produced by Mycobacterium leprae and in the synovial fluid of joints affected by rheumatoid arthritis, raising the possibility that this subset of T lymphocytes may play a role in control of infectious processes and in autoimmune diseases. We report the identification of a TCR-gamma delta T cell clone isolated from a HSV-infected mouse that recognizes glycoprotein I of HSV type 1. Clone recognition of glycoprotein I does not appear to require the expression of MHC class I or class II gene products. These data suggest that TCR-gamma delta lymphocytes may play an important role in the immune response to viral infections.
Animals↗
Cytolytic T lymphocytes: an overview of their characteristics.
Cloned T cells have been useful for assessing the lytic potential of distinct T cell subsets and for determining the relative contribution of different effector mechanism involved in the lytic process. Alloreactive CD8+ murine T cell clones and cloned murine CD4+ TH1 and TH2 T cells reactive with nominal antigen (ovalbumin) lysed nucleated target cells bearing antigen or coated with anti-CD3 monoclonal antibody in a short term 51Cr-release assay. These clones were also evaluated for their ability to lyse efficiently sheep erythrocyte (SRBC) target cells coated with anti-CD3 mAb by a mechanism (presumably involving membrane damage) that does not involve nuclear degradation. Three patterns of lysis were observed: CD8+ and some CD4+ TH2 effector cells lysed efficiently nucleated target cells and anucleated SRBC coated with anti-CD3 mAb. However, CD4+ TH1 (and a few TH2) T cells which lysed nucleated target cells bearing antigen or coated with anti-CD3 mAb did not lyse efficiently the SRBC coated with anti-CD3 mAb. One CD4 bearing TH2 cell failed to lyse efficiently either nucleated target cells or anucleated SRBC coated with anti-CD3 mAb. These results indicate that both TH1 and TH2 clones have lytic capabilities. Furthermore, they suggest that some but not all TH2 murine T cell clones have lytic characteristics similar to those of conventional CD8+ CTL. However, it is not certain how these patterns of lysis of target cells in vitro relates to the capacity of CTL to lyse such target cells in vivo.
Animals↗
Some cloned murine CD4+ T cells recognize H-2Ld class I MHC determinants directly. Other cloned CD4+ T cells recognize H-2Ld class I MHC determinants in the context of class II MHC molecules.
Murine T lymphocytes recognize nominal Ag presented by class I or class II MHC molecules. Most CD8+ T cells recognize Ag presented in the context of class I molecules, whereas most CD4+ cells recognize Ag associated with class II molecules. However, it has been shown that a proportion of T cells recognizing class I alloantigens express CD4 surface molecules. Furthermore, CD4+ T cells are sufficient for the rejection of H-2Kbm10 and H-2Kbm11 class I disparate skin grafts. It has been suggested that the CD4 component of an anti-class I response can be ascribed to T cells recognizing class I determinants in the context of class II MHC products. To examine the specificity and effector functions of class I-specific HTL, CD4+ T cells were stimulated with APC that differed from them at a class I locus. Specifically, a MLC was prepared involving an allogeneic difference only at the Ld region. CD4+ clones were derived by limiting dilution of bulk MLC cells. Two clones have been studied in detail. The CD4+ clone 46.2 produced IL-2, IL-3, and IFN-gamma when stimulated with anti-CD3 mAb, whereas the CD4+ clone 93.1 secreted IL-4 in addition to IL-2, IL-3, and IFN-gamma. Cloned 46.2 cells recognized H-2Ld directly, whereas recognition of Ld by 93.1 apparently was restricted by class II MHC molecules. Furthermore, cytolysis by both clones 46.2 and 93.1 was inhibited by the anti-CD4 mAb GK1.5. These results demonstrate that CD4+ T cells can respond to a class I difference and that a proportion of CD4+ T cells can recognize class I MHC determinants directly as well as in the context of class II MHC molecules.
Animals↗
Mechanisms of lysis by cytotoxic T lymphocyte clones. Lytic activity and gene expression in cloned antigen-specific CD4+ and CD8+ T lymphocytes.
Cloned murine Th having properties of either Th1 or Th2 cells as well as CD8+ CTL were tested for the capacity to lyse: 1) nucleated target cells bearing Ag or coated with anti-CD3 mAb, or 2) SRBC target cells coated with anti-CD3 mAb in a short term 51Cr-release assay. The lysis of SRBC occurs by a mechanism that does not involve nuclear degradation but presumably does involve membrane damage. Three patterns were observed: CTL and some Th2 cells lysed efficiently nucleated target cells and SRBC coated with anti-CD3 mAb. Th1 and some Th2 T cells lysed nucleated target cells but did not lyse efficiently the SRBC coated with anti-CD3 mAb. Finally, some Th2 cells failed to lyse efficiently either nucleated or SRBC targets. We also examined these clones for their expression of N-alpha-benzyloxycarbonyl-L-lysin thiobenzyl esterase activity, and for the expression of perforin or CTLA-1 (granzyme B) mRNA. Total N-alpha-benzyloxycarbonyl-L-lysin thiobenzyl esterase activity expressed by CTL and Th2 clones tended to be higher than that of Th1 cells. Perforin mRNA and CTLA-1 mRNA were readily detectable in CTL and some Th2 clones. Expression of perforin and CLTA-1 mRNA correlated well with the capacity of these clones to lyse SRBC coated with anti-CD3 mAb. Our results show that some but not all Th2 clones have lytic characteristics similar to those of CD8+ CTL. Two mechanisms appear to contribute to their lytic process, one mechanism of lysis involves membrane damage that correlates with the expression of perforin mRNA; a second mechanism involves the induction of DNA degradation in the target cells. In contrast, some CD4+ effector cells appear to lack the capacity to lyse efficiently via the mechanism involving membrane damage and may only have the lytic activity associated with the capacity to induce DNA degradation.
Animals↗
Murine Th1 and Th2 clones proliferate optimally in response to distinct antigen-presenting cell populations.
We recently have devised a method for the derivation of OVA-specific Th1 and Th2 clones from the same primed lymph node cell preparation. Using a panel of such cells, we have examined the ability of distinct APC populations to stimulate proliferation of Th1 and Th2 clones. Both subsets proliferated well in response to OVA in the presence of whole spleen cells. However, purified B cells stimulated optimal proliferation of Th2 clones, whereas adherent cells stimulated optimal proliferation of Th1 clones. The proliferative response of Th2 cells stimulated with spleen cells irradiated with 3300 rad was dramatically less than that observed in response to spleen cells treated with 1000 rad; Th1 clones responded similarly to spleen cells exposed to either irradiation dose. Differential activation of Th1 and Th2 clones did not correlate with MHC-restricting element, or susceptibility to inhibition by mAb directed against CD4 or LFA-1. Lymphokine production by each subset still occurred under conditions of suboptimal proliferation, suggesting that the appropriate Ag processing and presentation events had transpired. The same pattern of response was observed using a specific OVA peptide that does not require processing, suggesting that differential responsiveness of Th1 and Th2 clones to different APC populations is not a result of defective Ag processing. Neither rIL-1 nor rIL-6 restored optimal proliferation of either subset. Our results suggest that unique cofactors are necessary for the optimal proliferation of Th1 and Th2 clones, and that these cofactors are produced by specialized APC populations.
Animals↗
Differential activation of murine TH1 and TH2 clones.
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Animals↗
Herpes simplex virus glycoprotein D is recognized as antigen by CD4+ and CD8+ T lymphocytes from infected mice. Characterization of T cell clones.
Several previous reports have described the surprising inability to detect murine CTL specific for glycoprotein D (gD), one of the important protective immunogens of HSV. Using slight variations of published procedures, we were able to show that the immune response to HSV in infected mice includes the generation of CTL specific for gD. C3H/OuJ (H-2k) mice were infected by injection in the hind footpads with purified HSV-1. Lymphocytes from draining lymph nodes were then isolated and shown to proliferate in response to, and to kill, transformed fibroblasts (H-2k) expressing HSV-1 gD. Two gD-specific T cell clones were isolated. One clone, designated CGD1, was shwon to be CD8+. This clone recognizes HSV-1 gD, but not HSV-2 gD, in the context of class I MHC molecules and kills the appropriate MHC-matched fibroblasts expressing HSV-1 gD. Unusual features of this cytolytic clone include augmentation by IL-4 of proliferative responses to Ag, inhibition of its lytic activity by a mAb specific for Thy-1 and recognition of infected fibroblasts in preference to infected lymphoblasts. The other clone, designated CGD3, was shown to be CD4+. This clone recognizes both HSV-1 gD and HSV-2 gD in the context of class II MHC molecules and has cytolytic potential.
Animals↗
CD4+ murine T cell clones that express high levels of immunoglobulin binding belong to the interleukin 4-producing T helper cell type 2 subset.
A panel of 20 murine CD4+ clones was examined for the presence of surface membrane receptors for IgA, IgM, IgD, IgE, and IgG. High level expression of multiple Fc receptors (FcRs) was found on all Th2 clones. FcR expression was low or undetected on the Th1 clones. The preferential expression of FcR on activated Th2 cells suggests potential mechanisms for immunoregulatory interactions with B cells.
Animals↗