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While it was known by the 1960s that lymphocytes mediated adaptive immunity, it was unknown how antigens stimulated lymphocytes. Between 1967 and 1973, we reported that a rare cell type in murine spleen cells took up antigen and were obligatory for T cell dependent and independent antibody responses. We referred to them as A cells or the third cell type. In 1973, Ralph Steinman and Zanvil Cohn described a rare cell type in murine spleen cells which was phagocytic but had dendrite like protrusions; they named them dendritic cells (DCs). In 1978, Steinman reported that DC were required for mixed lymphocyte reactions. From that time until recent death, Ralph Steinman pursued relentlessly in his laboratory and through collaborations around the world the role and function of DC in immunity. In passing, using a monoclonal antibody supplied by Steinman, we showed that A cells were the same as DC., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Spectacular success in preventing renal allograft rejection in rats was obtained over 40 yr ago using only the reactants of the response: donor-type antigen and homologous antiserum directed against donor-type antigen. Tolerance was antigen specific and sustained by persistent antigen of the graft. The model has never been tested rigorously in a large species, though the rationale for why the procedures should work applies across species including humans. Confirming the results in a large species would have profound impact on research for treating multiple immune mediated diseases, in addition to providing a way for treating some transplant recipients. This is a propitious time to confirm the applicability to larger species. If successful, only the lack of imagination limits the potential impact., (© 2010 John Wiley & Sons A/S.)

This unit describes protocols for the generation of polyclonal T(H)1 and T(H)2 cell lines from naive CD4(+) T cells as well as for generation of antigen-specific cell lines from TCR-transgenic mice and antigen-specific T cell clones from primed mice. Also described are methods for the preparation and maintenance of alloreactive murine helper T (T(H)) lymphocyte and cytotoxic T lymphocyte (CTL) clones using the limiting dilution technique, as well as derivation of T(H) clones reactive with soluble protein antigens, including a method for the selection of either T(H)1 or T(H)2 lymphocyte subsets. These two subsets of T(H) cells exhibit helper function in different ways and can be distinguished by the patterns of cytokines they synthesize. Support protocols describe a micromanipulation method for cloning T cells and a roadmap for using protocols published elsewhere in this series to assess cytokine production by T cell clones and lines.

Mycoplasma contamination is a serious and frequent problem in the culture laboratory. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis rests on the detection of adenosine phosphorylase secretion by infected cell lines. This appendix describes how to test for mycoplasma contamination, and also presents methods for antibiotic treatment of infected cultures.

Using a TCR transgenic mouse bred onto a recombinase-activating gene-2-deficient background, we have examined the influence of B7.1 and B7.2 on activation of naive, CD8+ T cells in vitro. We found that B7.1 was a more potent costimulus than B7.2 for induction of proliferation and IL-2 production by naive CD8+ T cells. This difference appeared to be quantitative in nature, as determined using transfectants expressing various defined levels of B7.1 or B7.2, or using purified B7.1 or B7.2 fusion proteins. In contrast to the quantitative differences seen in stimulation of naive T cells, B7.1 and B7.2 were comparable in their ability to costimulate responses in T cells previously primed in vitro. In addition, primed, but not naive, T cells were capable of proliferating and producing IL-2 in response to a TCR stimulus alone, apparently in the absence of B7 costimulation. Lastly, we found that B7.1 and B7.2 were equivalently capable of driving differentiation of naive CD8+ T cells into an IL-4-producing phenotype when exogenous IL-4 was added to the primary culture or to an IFN-gamma-producing phenotype in the presence of IL-12. These results indicate that signals generated by B7.1 and B7.2 are qualitatively similar, but that B7.1 is quantitatively stronger than B7.2. Further, our results indicate that the activation state of the responding T cell may influence the efficiency with which the T cell can respond to a costimulatory signal provided by either B7.1 or B7.2.

CD28 signaling is critical for IL-2 production by established Th1 clones, but CD28 does not appear to play a role in the activation of established Th2 clones. To determine the role of CD28 in the generation of polarized T cells, clones were derived using cells from CD28-deficient (CD28-/- mice, which had been bred with mice that express the DO11.10 transgene, a CD4+ TCR-alphabeta receptor that recognizes OVA peptide 323-339 bound to I-Ad. Most T cell clones derived from CD28+/+ mice survived multiple stimulations, while T cell clones derived from CD28-/- mice survived only if they were derived initially in the presence of IL-4 or both IL-2 and IL-4. Signaling through the CD28 molecule did not appear to be important in the initial activation of T cell clones, as the precursor frequency of clones derived from normal (CD28+/+) and CD28-/- mice was similar. Primary stimulation in the presence of IL-4 increased cell number and viability of both CD28+/+ and CD28-/- T cells in primary culture. However, the survival of CD28-/- cells is more dependent on IL-4 than is the survival of CD28+/+ cells. The continued presence of anti-IL-4 mAb dramatically decreased the number of viable cells in the CD28-/- cultures but had little effect on the viability of the CD28+/+ clones. Thus, initial culture with IL-4 allows the isolation of CD28-/- T cell clones that produce IL-4. In these clones, IL-4 acts as both an autocrine growth and survival factor.

CD28 ligation augments TCR-mediated proliferation, IL-2 production, and T cell survival. However, the role of CD28 costimulation in T cell differentiation remains controversial. To address this issue, CD28+ and CD28-deficient TCR alphabeta transgenic (Tg) mice were used to examine cytokine production by T cells following antigenic stimulation. Increasing CD28 ligation resulted in increased production of IL-4 and IL-5, consistent with differentiation toward a Th2 phenotype, in both CD4+ TCR Tg T cells and CD8+ TCR Tg T cells. The same result was obtained with CD4+ TCR Tg mice bred to RAG2-deficient mice, indicating that the Th2 differentiation observed with increased CD28 ligation was not due to the presence of memory T cells. Although CD28 costimulation is an essential factor regulating IL-2 synthesis, differentiation toward a Th2-like phenotype by CD28 ligation was not an indirect effect of enhanced IL-2 production. In contrast, blockade of IL-4 during the primary cultures of the T cells resulted in a profound inability to produce Th2-type cytokines upon restimulation. The critical role of IL-4 was confirmed by the finding that CD28-deficient TCR alphabeta Tg+ T cells cultured with rIL-4 differentiated into Th2-like T cells. Therefore, CD28 ligation promotes the production of Th2-type cytokines by naive murine T cells via an IL-4-dependent mechanism.

Stimulation of interleukin-2 producing T lymphocytes via the T cell receptor (TCR) complex in the absence of other costimulatory factor results paradoxically not in activation but in an unresponsive state termed clonal anergy. T cell anergy appears to be a mechanism by which potentially autoreactive T lymphocytes are inactivated in the periphery, thus maintaining tolerance to self antigens. The breakdown of such tolerance may result in autoimmune diseases. In contrast, induction of peripheral tolerance is the ultimate goal in organ transplantation and is a potential mechanism by which a growing tumor evades immune destruction. The anergic state is characterized by an inability to secrete interleukin-2 and proliferate following restimulation via the TCR even in the presence of constimulatory factors. Recent studies have demonstrated a specific block in Ras activation in anergic T lymphocytes. This defect is correlated with a failure to activate the downstream effectors Erk and Jnk and a lack of activation of the AP-1 transcription factor complex, offering a plausible mechanism for the inability to initiate interleukin-2 gene transcription in the anergic state.

Unlike TCR-alpha beta cells, TCR-gamma delta cells express a distinct member of the zeta family, the gamma-chain of Fc epsilon RI (Fc epsilon RI gamma) within the TCR complex. To study the role of the Fc epsilon RI gamma-chain in TCR-gamma delta cells, a TCR-gamma delta transgenic mouse (G8) has been crossed with CD3 zeta-chain-deficient mice (G8.zeta-/-). Thy-1+ spleen and lymph node cells of these animals expressed low levels of CD3/TCR. These results suggested that the zeta-chain is required for effective TCR transport to the cell surface. In contrast, intraepithelial TCR-gamma delta cells of G8.zeta-/- mice expressed high levels of TCR. Immunoprecipitation with anti-CD3 showed that Fc epsilon RI gamma-chains were associated with the TCR complex in T cells isolated from zeta-deficient mice. Although the Fc epsilon RI gamma-expressing T cells proliferated in response to stimulation by TCR-specific Abs including anti-CD3 epsilon, anti-pan gamma delta, and anti-V gamma 2 mAb, the G8.zeta-/- T cells did not respond to the G8-specific Ag (T10b), anti-Thy-1 mAb, or Con A. The unresponsiveness to the Ag was not due to the reduced TCR expression, because intraepithelial TCR-gamma delta cells from the zeta-deficient mice did not respond to Ag. The inability of the G8.zeta-/- T cells to respond to Ag could not be overcome by providing an anti-CD28 costimulatory signal or by adding exogenous rIL-2. Taken together, our data suggest that the Fc epsilon RI gamma-chain associates with the TCR-gamma delta complex in the absence of the zeta-chain, but it is not able to substitute for the zeta-chain for effective transport of TCR to the cell surface or functional responses to Ag.

T cell anergy is a state of functional unresponsiveness characterized by the inability to produce interleukin-2 (IL-2) upon T cell receptor stimulation. The mitogen-activated protein kinases ERK-1 and ERK-2 and the guanosine triphosphate-binding protein p21ras were found to remain unactivated upon stimulation of anergic murine T helper cell 1 clones. The inability to activate the Ras pathway did not result from a defect in association among Shc, Grb-2, and murine Son of Sevenless, nor from a defect in their tyrosine phosphorylation. This block in Ras activation may lead to defective transactivation at activator protein 1 sites in anergic cells and may enable T cells to shut down IL-2 production selectively during anergy.

The OVA-reactive CD4+ Th1 clones and alloreactive CD8+ clones derived from wild-type or fyn-/- mice serve as model systems which have allowed us to investigate several aspects of the molecular events associated with T cell-mediated cytotoxicity, including 1) the differential utilization of two distinct cytolytic pathways by CD4+ Th1 clones and CD8+ CTL, 2) a comparison of the pathways of lysis induced by stimulation of the TCR or by alternative stimuli, 3) the requirement of Fyn for derivation of antigen-specific T-cell clones having properties of CD4+ Th1 and CD8+ CTL cells 4) the differential requirement of Fyn in the induction of responses by TCR and the alternative stimuli. Stimulation through the TCR, either by APC bearing relevant antigen or by immobilized anti-CD3 mAb, resulted in comparable levels of target cell lysis by clones from both wild-type and fyn-/- mice. These clones also utilize the Fas pathway to lyse target cells. Thus, Fyn does not appear to be required for expression of the Fas pathway when triggered through the TCR. In contrast, lysis of target cells by T-cell clones lacking Fyn was deficient when stimulated through Thy-1 or Ly-6C (using mAb) or with Con A or phorbol ester as compared to clones derived from wild-type mice. The basis for the defect in response to stimulation through the GPI-linked molecules appears to be a signaling defect which affects all of the functional responses we measured, while the defect in response to Con A stimulation appears to affect lysis but not lymphokine production. Thus, Fyn expression is selectively required for efficient activation of the Fas pathway of lysis through Thy-1, Ly-6C, and by Con A or phorbol ester in these T-cell clones. CD8+ clones derived from fyn-/- mutant mice, like clones derived from wild-type mice, display antigen-specific lysis, and appear to express perforin message and perforin protein. A Ca(++)-dependent (presumably perforin/exocytosis) component and Fas component of lysis was detected in CD8+ clones derived from fyn-/- mutant mice. Thus, Fyn is not required for expression of these components of antigen specific lysis by CD8+ alloreactive CTL clones. It appears that CD8+ clones that use multiple lytic mechanisms may selectively employ the perforin or Fas-based pathway depending on properties of the target cell or stimulus.(ABSTRACT TRUNCATED AT 400 WORDS)

Several alterations in T cell receptor-associated signal transduction have been observed following induction of anergy of T helper type 1 (Th1) clones, including a modified intracellular free calcium ([Ca2+]i) response and increased kinase activity associated with the protein tyrosine kinase p59fyn. In the current study, we demonstrate that, although the kinetics of acquisition of both of these signaling alterations correlated with the generation of anergy, a normal calcium response returned within 48 h after removal from the anergizing stimulus, whereas the increased p59fyn activity persisted and the cells remained hyporesponsive. Generation of both the anergic state and the increased p59fyn activity was prevented in the presence of calcium-free medium, cycloheximide (CHX), or cyclosporin A (CsA), and could be mimicked by the calcium ionophore ionomycin. In contrast, the altered calcium response was inhibited by stimulation in the presence of calcium-free medium or CsA, but not CHX. Thus, surprisingly, these data suggest that a chronic elevation of [Ca2+]i is proximal to and necessary for the increase in p59fyn-associated kinase activity observed in anergic Th1 clones. Increased p59fyn activity, but not the altered calcium response, correlates with maintenance of the anergic state.

The protein tyrosine kinase Fyn has been shown to be involved in signal transduction through the TCR and the glycosyl-phosphatidylinositol-linked surface molecule Thy-1 expressed on T cells. In this study, we examine the requirement for Fyn expression in signaling through the TCR or Thy-1 using a panel of Ag-specific T cell clones derived from fyn-/- mutant mice. These clones do not express normal Fyn protein, as measured by immune-complex kinase reaction using anti-Fyn Ab. Stimulation through the TCR, either by APC bearing relevant Ag or by immobilized anti-CD3 mAb, resulted in comparable levels of proliferation, lymphokine production, and cytolysis by clones from both wild-type and fyn-/- mice. In contrast, stimulation through Thy-1, using soluble (or cross-linked) anti-Thy-1 mAb, was deficient, as measured by these responses. Thus, Fyn expression is selectively required for functional activation through Thy-1 in these T cell clones.

Interactions between CD4+ T cells and B cells are mediated by both soluble factors and cell surface molecules. The Ag-independent interaction between the CD40 ligand, expressed on activated T cells, and its CD40 receptor, expressed on B cells, enhances B cell proliferation in response to IL-4 stimulation. The expression of the CD40 ligand is induced on CD4+ T cells by stimulation with Ag-pulsed APC or mitogens. Here, we show that at least some IL-4-producing murine CD8+ T cell clones can be induced to express the CD40 ligand when stimulated with anti-CD3 mAb. Additionally, such activated CD8+ IL-4-producing clones potentiate the proliferative response of small resting B cells to IL-4 and induce Ig secretion by small resting B cells to IL-4 and IL-5. Proliferation of small resting B cells cultured with IL-4 in the presence of activated IL-4-producing CD8+ murine T cell clones appeared to be mediated by the expression of the CD40 ligand on the T cell because an anti-CD40 ligand mAb inhibited this proliferative response. A conventional murine CD8+ CTL clone, which did not produce IL-4 or express CD40 ligand upon activation, did not potentiate proliferation of small resting B cells exposed to IL-4. Thus, under some circumstances, CD8+ T cells that are able to express CD40 ligand may be able to provide B cell help.

Patterns of cytokine secretion and functional differences distinguish T lymphocyte subsets. T lymphocyte subsets are also regulated differentially. Most established CD8+ lymphocyte clones secrete gamma-interferon (IFN-gamma) but not interleukin 2 (IL-2) or IL-4. Using murine T cells which express a transgenic, antigen-specific alpha/beta T cell receptor (TCR) specific for L(d) class I major histocompatibility complex antigen, we have found that CD8+ lymphocytes can be divided into functional subsets. Freshly isolated CD8+ T cells are not cytolytic, do not proliferate and do not proliferate and do not secrete cytokines. Stimulation of TCR alone does not induce cytokine secretion, but cells become responsive to exogenous IL-2 or IL-4. Stimulation of CD28 together with TCR induces secretion of IL-2 and IFN-gamma, and cells proliferate without exogenous cytokines. Proliferation is necessary for the development of cytolytic activity. If IL-4 is present during initial stimulation, IL-4 is secreted following restimulation. Upon stimulation, some IL-4-producing murine CD8+ T cell clones express CD40 ligand (CD40L), and they potentiate proliferation and immunoglobulin secretion by small resting B cells. Thus, the CD8+ T cell subsets T cytotoxic 1 (Tc1) and Tc2 are analogous to CD4+ T helper 1 (Th1) and Th2. IL-2 production by naive CD8+ cells requires co-stimulation. IL-4 production by CD8+ T cells requires the presence of IL-4 during initial stimulation. Some IL-4-producing CD8+ T cells express CD40L following TCR stimulation and provide help for B cells.

IL-4 has been shown to be involved in the early stages of B cell maturation. Changes induced by IL-4 include cell enlargement, increased viability, and increased MHC class II expression. However IL-4 alone does not induce B cell activation as defined by proliferation, lymphokine production, or Ig class switching. In this study, we demonstrate that incubation with IL-4 enhances the ability of small splenic murine B cells, normally poor stimulators of murine Th1 clones, to stimulate lymphokine production and proliferation by Th1 clones. Moreover, small resting B cells induce anergy, whereas IL-4-treated B cells do not. IL-4-treated B cells were found to express both B7 (B7-1) and a second ligand for CTLA4Ig (B7-2). Although IL-4 induces both B7-1 and B7-2, the kinetics of expression of these molecules are different: B7-2 is detected by 6 h, whereas B7-1 is not detectable until 48 h. In addition, only CTLA4Ig fully blocks IL-4 induced costimulatory activity; a mAb to B7-1 does not. Thus, these results suggest that IL-4 may function indirectly as a costimulatory factor by inducing costimulatory molecules on resting B cells. Additionally, these findings support our previous findings that an alternative ligand for CD28 and CTLA4 is important in providing costimulation.

TCR-gamma delta cells, a T cell subset present in the epithelial and lymphoid tissues, have been implicated in viral and bacterial infections. We have identified a TCR-gamma delta clone (TgI4.4) that, unlike TCR-alpha beta cells, recognizes a herpes simplex virus type 1 transmembrane glycoprotein, gI, in an MHC class I- and class II-independent fashion. The TCR of TgI4.4 is composed of rearranged V delta 8 (a V alpha 2 family member) and V gamma 1.2 variable genes, a heterodimeric pair not previously described. Furthermore, anti-V alpha 2 mAbs are sufficient to block recognition of the gI ligand. Strikingly, anti-gI Abs also are capable of blocking recognition, a phenomena that is very rare in TCR-alpha beta Ag recognition. Therefore, to dissect the mechanism involved in this unique form of Ag recognition, we constructed a mutant of gI, gIt, that lacks cell surface expression upon transfection into APCs. This form of gI was not sufficient for Ag presentation. In contrast, wild-type gI expressed in the Ag-processing mutant cell, RMA-S, is recognized by TgI4.4, suggesting that gI presentation occurs independently of classical Ag-processing pathways. In fact, through the use of a soluble recombinant gI molecule, gI-Ig, we show that TgI4.4 can recognize whole, unprocessed gI protein in the absence of any APCs. These results suggest that there exist alternate and novel forms of TCR Ag recognition, and that the TCR-gamma delta clone, TgI4.4, may represent a novel T cell subset that, during pathogenic challenge, may respond directly to Ags on the surfaces of bacteria and viruses.

Mature CD4+ helper T lymphocytes have been categorized into two major functional phenotypes, TH1 and TH2, which produce distinct arrays of lymphokines and which are thought to arise from a pluripotential precursor cell termed TH0. Clonal anergy can be induced in TH1 clones by stimulating via the T cell receptor (TCR) complex in the absence of a costimulator molecule; however, anergy has been difficult to demonstrate in TH2 clones. We show here that treatment of cloned TH0 lines with anergizing stimuli results in the selective loss of TH1 characteristics and retention of a TH2 phenotype. Treated cells exhibit a substantial reduction in interleukin 2 (IL-2) production and antigen-specific cytolytic activity, but retain comparable IL-4 and IL-5 production in response to restimulation via the TCR complex. TH0 clones exposed to anergizing stimuli also increase in size, thus morphologically resembling TH2 cells. The signaling characteristics of these cells also are altered, in that they exhibit an elevated basal level of intracellular free calcium which fails to increase significantly with subsequent restimulation, reminiscent of the signaling characteristics of TH2 cells. "Anergized" TH0 clones thus share several functional, morphologic, and physiologic properties with cells of the TH2 phenotype, suggesting that TH2 cells may arise when TH0 cells are stimulated via the TCR complex in the absence of a putative costimulator molecule.

Full activation of TH1 helper T lymphocytes requires ligation of the specific T-cell antigen receptor (TCR) and a second signal provided by costimulator molecule(s) expressed on particular antigen-presenting cells. Stimulation via the TCR complex alone generates a subsequent unresponsive state characterized by an inability to produce interleukin 2. We report here that such anergic cells exhibit multiple alterations in TCR-associated signaling. The basal levels of intracellular free calcium and phosphatidylinositol 1,4,5-trisphosphate are elevated in anergic cells, and the levels fail to increase significantly upon subsequent restimulation. Examination of phospholipase C-gamma 1 reveals evidence for post-translational modification, correlating with increased tyrosine phosphorylation of the molecule. Tyrosine phosphorylation of additional substrates identified from whole-cell lysates also is altered compared to untreated cells, suggesting a modification in net tyrosine kinase activity. Although the level of kinase activity present in TCR/CD3 or Lck immunoprecipitates is modestly altered after induction of anergy, there is a dramatic increase in specific Fyn-associated tyrosine kinase activity in anergic cells and increased phosphorylation of a 110-kDa protein that is coimmunoprecipitated with Fyn. These results are consistent with a model in which anergic TH1 lymphocytes display a fundamental alteration in TCR-mediated tyrosine kinase activity, associated with changes in phospholipase C-gamma 1, inositol phosphates, and intracellular free calcium.

Cytolytic effector function fails to develop if proliferation of allospecific cytolytic T lymphocyte precursors is inhibited, but the requirements for generation of cytolytic activity have not been fully defined. In contrast, the cytolytic effector function of cytolytic T lymphocyte clones does not change during the cell cycle, and the level of cytolytic activity is independent of cellular proliferation. The requirement for proliferation by primary responding populations may reflect the need for clonal expansion of a few inherently cytolytic effector cells in order to reach a threshold number which can readily be detected in conventional cytolytic assays. Alternatively, proliferation may be required for cytolytic T lymphocyte precursors to differentiate into mature, functional cytolytic cells. Using CD8+ T cells which express an antigen-specific transgenic alpha/beta T cell receptor, we have studied the requirements for acquisition of cytolytic capacity. Stimulation of the T cell receptor alone appears to be sufficient to render naive, CD8+ transgenic T cells sensitive to the growth effects of interleukin-2 (IL-2), and in some circumstances to interleukin-4 (IL-4), but not to induce either lymphokine production or cytolytic activity. Costimulatory molecules expressed by allogenic stimulating cells appear to be required for lymphokine production, and CD8+ transgenic T cells initially appear to secrete only IL-2 and interferon-gamma. Stimulation of the T cell receptor of naive, CD8+ transgenic T cells appears to induce cytolytic activity only if cell proliferation occurs, either in response to IL-2 produced by the stimulated cells themselves when costimulatory molecules are present, or to IL-2 or IL-4 from exogenous sources if costimulatory molecules are absent.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Ly-6C is a differentiation antigen that distinguishes T-lymphocyte subsets. In concordance with previous results, splenocytes from NOD mice do not express the epitope recognized by anti-Ly-6C monoclonal antibodies (MoAbs), including MoAb HK1.4 in this study, and cannot be stimulated to proliferate in response to HK1.4. However, when splenocytes from NOD mice were stimulated in vitro with the anti-CD3 MoAb 145-2C11, T lymphocytes expressing Ly-6C were detected after 48 h of stimulation, with as many as 25% of lymphocytes expressing this antigen with prolonged passage in culture. Most of the cells expressing Ly-6C were Thy-1.2+, CD4+, and CD8- and proliferated after stimulation with HK1.4. To further understand the failure of NOD splenocytes to express Ly-6C, freshly isolated cells were stimulated with alpha/beta-interferon (IFN-alpha/beta) and IFN-gamma. Although these lymphokines induced expression of Ly-6A and Ly-6C in splenocytes from C57BL/6J mice and Ly-6A in NOD cells, Ly-6C was not induced on NOD cells. Because Ly-6C expression on splenocytes was a marker of activation via the CD3 T-lymphocyte receptor complex, we also examined expression of Ly-6C on T lymphocytes within islets showing insulitis in vivo. Lymphocytes that were Ly-6C+ were identified within islets on histological sections of pancreas, whereas Ly-6C+ cells in the spleen from the same mouse could not be detected. Our findings imply functional abnormality in expression of Ly-6C in NOD mice.(ABSTRACT TRUNCATED AT 250 WORDS)

We have reported recently that high concentrations of anti-CD3 mAb inhibited IL-2-dependent proliferation of TH1 but not TH2 clones. The selective inhibitory effect on TH1 clones suggested that the two helper T lymphocyte subsets might utilize different TCR-associated signal transduction mechanisms. In the present study, we demonstrate that this distinction was not due to a gross difference in the level of TCR expression by TH1 and TH2 clones. Inhibition of TH1 proliferation by anti-CD3 mAb appeared to depend on calcium for maximal effect, suggesting that a substantial elevation of intracellular free calcium concentration ([Ca2+]i) might not occur after ligation of the TCR complex of TH2 clones. Calcium ionophore inhibited IL-2-dependent proliferation of both subsets, suggesting that receptor/ligand systems which stimulate elevated [Ca2+]i would be expected to inhibit proliferation. Although elevated [Ca2+]i and generation of inositol phosphates were readily detected in TH1 clones, these second messengers were not detected following stimulation of TH2 clones via the TCR complex. In addition, lymphokine production by TH1 clones was more sensitive to inhibition by cholera toxin, 8-bromoadenosine 3':5'-cyclic monophosphate, and cyclosporin A than was lymphokine production by TH2 clones. Collectively, these results suggest that TH1 and TH2 clones utilize distinct TCR-associated signal transduction mechanisms for lymphokine gene expression. The difference in signaling mechanisms suggests a potential pharmacologic target for intervention in situations where inappropriate activation of TH1 or TH2 cells occurs in vivo.

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.

We have demonstrated recently that rIFN-gamma inhibits the proliferation of murine bone marrow cells stimulated with rIL-3 or recombinant granulocyte-macrophage (GM)-CSF. In light of this finding, three murine CD8+ CTL clones whose supernatants had been shown previously not to contain detectable levels of CSF activity but which contained a relatively high level of IFN-gamma were reassessed for their ability to secrete IL-3 and GM-CSF. Supernatants from CTL clones activated with anti-CD3 mAb failed to stimulate the IL-3-dependent cell line FDCP1. However, these supernatants were indeed able to stimulate the proliferation of FDCP1 cells if anti-IFN-gamma mAb was present. This stimulatory activity was specifically neutralized by anti-IL-3 mAb. Supernatants from two of the three clones stimulated the proliferation of a GM-CSF-responsive HT-2 cell line, and this activity was neutralized by anti-GM-CSF antibody. The otherwise modest ability of CTL supernatants to stimulate the proliferation of fresh bone marrow cells was augmented considerably in the presence of anti-IFN-gamma mAb, and this activity was appropriately blocked by anti-IL-3 and anti-GM-CSF antibodies. mRNA for IL-3 and GM-CSF, as well as for IFN-gamma and TNF-alpha, was detected in cells that secreted those lymphokines, and the time course of appearance of each mRNA correlated with secretion of the appropriate lymphokine activity. However, the time course of mRNA accumulation for each lymphokine was distinct, the order of expression of these genes apparently being TNF-alpha, then IFN-gamma and GM-CSF, and finally IL-3. Our results emphasize that potential interactions among lymphokines must be considered when interpreting data obtained from lymphokine bioassays and suggest an immunoregulatory role for CTL through the secretion of several of the same lymphokines produced by HTL.