Indoleamine 2,3-dioxygenase, an emerging target for anti-cancer therapy.

The inability of the host immune system to control tumor growth appears to result from dominant mechanisms of immune suppression that prevent the immune system from effectively responding in a way that consistently results in tumor rejection. Among the many possible mediators of tumoral immune escape, the immunoregulatory enzyme, indoleamine 2,3-dioxygenase (IDO), has recently gained considerable attention. IDO is a heme-containing, monomeric oxidoreductase that catalyzes the first and rate-limiting step in the degradation of the essential amino acid tryptophan to N formyl-kynurenine. Tryptophan depletion as well as the accumulation of its metabolites results in a strongly inhibitory effect on the development of immune responses by blocking T cell activation, inducing T cell apoptosis and promoting the differentiation of naïve T cells into cells with a regulatory phenotype (T(regs)). Recent data obtained from preclinical tumor models demonstrate that IDO inhibition can significantly enhance the antitumor activity of various chemotherapeutic and immunotherapeutic agents. These results, coupled with data showing that increased IDO expression is an independent prognostic variable for reduced overall survival in cancer patients, suggest that IDO inhibition may represent an effective strategy to treat malignancies, either alone or in combination with chemotherapeutics or other immune based therapies. This review will focus on the role of IDO as a mediator of peripheral immune tolerance, evidence that IDO becomes dysregulated in human cancers, and the latest progress on the development of IDO inhibitors as a novel anti-cancer therapy.

ADAM-mediated amphiregulin shedding and EGFR transactivation.

INTRODUCTION: The ectodomain shedding of epidermal growth factor receptor (EGFR) ligands, such as amphiregulin (AREG), by ADAMs (A Disintegrin And Metalloproteases) can be stimulated by G protein-coupled receptor (GPCR) agonists. Interactions between the CXCR4 GPCR and the CXCL12 chemokine have been shown to mediate gene transcription and cellular proliferation in non-transformed and transformed prostate epithelial cells, as well as motility/invasiveness in transformed cells. OBJECTIVES: In this report, we investigated the ability of CXCL12 to stimulate amphiregulin ectodomain shedding in non-transformed and transformed prostate epithelial cells that respond proliferatively to sub-nanomolar levels of CXCL12 and amphiregulin. MATERIALS AND METHODS: Non-transformed N15C6 and transformed PC3 prostate epithelial cells were assessed for amphiregulin shedding, ADAM activation, Src phosphorylation and EGFR activation using ELISA, immunoblot, and immunoprecipitation techniques, and for proliferation using cell counting after stimulation with CXCL12 or vehicle. RESULTS: The results of these studies identify CXCL12 as a novel inducer of amphiregulin ectodomain shedding and show that both basal and CXCL12-mediated amphiregulin shedding are ADAM10- and Src kinase-dependent in non-transformed N15C6 cells. In contrast, amphiregulin shedding is not amplified subsequent to stimulation with exogenous CXCL12, and is not reduced subsequent to metalloprotease- or Src kinase-inhibition, in highly aggressive PC3 prostate cancer cells. These data also show that CXCL12-mediated cellular proliferation requires EGFR transactivation in a Src- and ADAM-dependent manner in non-transformed prostate epithelial cells. However, these same mechanisms are dysfunctional in highly transformed prostate cancer cells, which secrete amphiregulin in an autocrine manner that cannot be repressed through metalloprotease- or Src kinase inhibition. CONCLUSION: These findings show that non-transformed and transformed prostate epithelial cells may employ different mechanisms to activate EGFR ligands and thereby utilize the EGFR axis to promote cellular proliferation.

Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-alpha.

The binding of tumor necrosis factor alpha (TNF-alpha) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-alpha to TNFRc1 (IC(50) = 50 nM) and also blocked TNF-stimulated phosphorylation of Ikappa-B in Ramos cells (IC(50) = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 microM. Detailed evaluation of this and related molecules revealed that compounds in this class are "photochemically enhanced" inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 microM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-alpha to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-alpha-TNFRc1 interaction.

Regulation of cyclooxygenase-2 induction in the mouse uterus during decidualization. An event of early pregnancy.

The infertility phenotype of cyclooxygenase-2 (Cox-2)-deficient female mice establishes the important role of Cox-2 in pregnancy. Cox-2 deficiency results in defective ovulation, fertilization, implantation, and decidualization; the latter of which can be restored in part by the prostacyclin analog carbaprostacyclin. Uterine Cox-2 expression during early pregnancy shows distinct localization and kinetics in the uterine luminal epithelium and underlying stromal cells, suggesting that expression is tightly regulated. Several intracellular signaling cascades including ERK, p38, and JNK are implicated in vitro as critical components of regulated Cox-2 expression in response to mitogens, growth factors, and cytokines. We investigated the involvement of these signaling pathways during Cox-2 induction in vivo by monitoring uterine kinase activity after intraluminal application of a deciduogenic stimulus. Our results show that the ERK and p38 pathways are activated in uterine preparations as early as 5-min post-stimulation. ERK activation was sustained for several hours with a return to baseline levels by 4 h. p38 activation was rapid with a peak at 5-min post-stimulation and returned to near baseline levels after 45 min. Systemic administration of a MEK inhibitor completely inhibited ERK activation, but did not affect early (2 h) luminal epithelial or late (24 h) stromal Cox-2 expression and only modestly affected decidualization. In contrast, administration of a p38 inhibitor modestly inhibited early Cox-2 expression in the luminal epithelium, while dramatically diminishing late stromal expression. In parallel, induced stromal peroxisomal proliferator activated receptor-delta (PPARdelta) expression is blunted by p38 inhibition. p38 inhibition also significantly inhibited decidualization. These results suggest that p38, but not ERK, activation is required for induced Cox-2 and PPARdelta expression during decidualization. In addition, inhibition of p38 led to decreased decidualization suggesting that an intracrine prostanoid pathway consisting of Cox-2, prostacyclin, and PPARdelta is required for maintenance of early pregnancy.

The extracellular signal-regulated kinase cascade is required for NMDA receptor-independent LTP in area CA1 but not area CA3 of the hippocampus.

Activation of extracellular signal-regulated kinase (ERK) has been shown to be necessary for NMDA receptor-dependent long-term potentiation (LTP). We studied the role of ERK in three forms of NMDA receptor-independent LTP: LTP induced by very high-frequency stimulation (200 Hz-LTP), LTP induced by the K(+) channel blocker tetraethylammonium (TEA) (TEA-LTP), and mossy fiber (MF) LTP (MF-LTP). We found that ERK was activated in area CA1 after the induction of both 200 Hz-LTP and TEA-LTP and that this activation required the influx of Ca(2+) through voltage-gated Ca(2+) channels. Inhibition of the ERK signaling cascade with either PD 098059 or U0126 prevented the induction of both 200 Hz-LTP and TEA-LTP in area CA1. In contrast, neither PD 098059 nor U0126 prevented MF-LTP in area CA3 induced by either brief or long trains of high-frequency stimulation. U0126 also did not prevent forskolin-induced potentiation in area CA3. However, incubation of slices with forskolin, an activator of the cAMP-dependent protein kinase (PKA) cascade, did result in increases in active ERK and cAMP response element-binding protein (CREB) phosphorylation in area CA3. The forskolin-induced increase in active ERK was inhibited by U0126, whereas the increase in CREB phosphorylation was not, which suggests that in area CA3 the PKA cascade is not coupled to CREB phosphorylation via ERK. Overall, our observations indicate that activation of the ERK signaling cascade is necessary for NMDA receptor-independent LTP in area CA1 but not in area CA3 and suggest a divergence in the signaling cascades underlying NMDA receptor-independent LTP in these hippocampal subregions.

Inhibition of MAP kinase kinase (MEK) results in an anti-inflammatory response in vivo.

The MAP kinase pathway has been well-characterized as a cascade of sequential protein phosphorylation events leading to the upregulation of a variety of genes in response to growth factors and mitogens. We are interested in the role of these kinases in inflammation and have thus examined their activity in vivo using TPA-induced ear edema in the mouse as a model of inflammation. We show that the activities of both ERK-1 and ERK-2 are upregulated in this model in response to TPA. Increased levels of ERK phosphorylation are measurable as early as 15 min poststimulation and reach a level 8-fold over controls at 4 h. In contrast, minimal activation of JNK or p38 is observed. Topical treatment of ears with the MEK inhibitor, U0126, prevents ERK phosphorylation and ear swelling in a dose-dependent manner in this model. These results suggest that the MEK/ERK pathway is important during an inflammatory response in vivo.

Inhibition of MAP kinase kinase prevents cytokine and prostaglandin E2 production in lipopolysaccharide-stimulated monocytes.

Activation of the extracellular signal-regulated kinase (ERK) pathway has been shown to occur in monocytes following stimulation with LPS. However, the importance of this event for monocyte function is not clear. To address this issue, we used the novel MAP/ERK kinase (MEK) inhibitor, U0126. Stimulation of monocytes with LPS resulted in activation of the mitogen-activated protein kinase (MAPK) family members ERK, Jun NH2-terminal kinase (JNK), and p38. Treatment of monocytes with LPS in the presence of U0126 blocked the activation of ERK1 and ERK2. However, the activation of Jun NH2-terminal kinase and p38 family members was not affected by the compound, confirming the selectivity of U0126. To examine the effects of MEK inhibition on monocyte function, we measured production of the cytokines IL-1, IL-8, and TNF, as well as PGE2. Monocytes treated with LPS in the presence of U0126 failed to release IL-1, IL-8, TNF, or PGE2. The failure to secrete IL-1 and TNF was due to decreased levels of mRNA. These results demonstrate that activation of MEK/ERK is critical for cytokine and PGE2 production by monocytes in response to LPS.

Rapid inhibition of interleukin-6 signaling and Stat3 activation mediated by mitogen-activated protein kinases.

Gene activation and cellular differentiation induced by interleukin-6 (IL-6) and transcription factor Stat3 are suppressed by several factors, including ionomycin, granulocyte/macrophage-colony-stimulating factor, and phorbol 12-myristate 13-acetate (PMA), that block IL-6-induced Stat3 activation. These inhibitory agents activate mitogen activated protein kinases (MAPKs), and thus the role of MAPKs in the mechanism of inhibition of Stat3 activation was investigated. Inhibition of IL-6-induced Stat3 activation by PMA and ionomycin was rapid (within 5 min) and did not require new RNA or protein synthesis. Inhibition of Stat3 DNA-binding activity and tyrosine phosphorylation by PMA, ionomycin, and granulocyte/macrophage-colony-stimulating factor was reversed when activation of the extracellular signal-regulated kinase (ERK) group of MAPKs was blocked by using specific kinase inhibitors. Expression of constitutively active MEK1, the kinase that activates ERKs, or overexpression of ERK2, but not JNK1, inhibited Stat3 activation. Inhibition of Stat3 correlated with suppression of IL-6-induction of a signal transducer and activator of transcription (STAT)-dependent reporter gene. In contrast to IL-6, activation of Stat3 by interferon-alpha was not inhibited. MEKs and ERKs inhibited IL-6 activation of Stat3 harboring a mutation at serine-727, the major site for serine phosphorylation, similar to inhibition of wild-type Stat3, and inhibited Janus kinases Jak1 and Jak2 upstream of Stat3 in the Jak-STAT-signaling pathway. These results demonstrate an ERK-mediated mechanism for inhibiting IL-6-induced Jak-STAT signaling that is rapid and inducible, and thus differs from previously described mechanisms for downmodulation of the Jak-STAT pathway. This inhibitory pathway provides a molecular mechanism for the antagonism of Stat3-mediated IL-6 activity by factors that activate ERKs.

Identification of a novel inhibitor of mitogen-activated protein kinase kinase.

The compound U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2, as U0126 shows little, if any, effect on the kinase activities of protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92, 7686-7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates, ATP and ERK. We further demonstrate that the two compounds bind to deltaN3-S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for deltaN3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the deltaN3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.

Competitive inhibition of MAP kinase activation by a peptide representing the alpha C helix of ERK.

On the basis of the crystal structure of the MEK substrate ERK, we have synthesized a 15 amino acid peptide representing the alpha C helix of human ERK1. We find this peptide to be an inhibitor of ERK phosphorylation by its upstream activator MEK. Circular dichroic spectroscopy indicates that the peptide has little secondary structure in aqueous buffer, but can readily adopt an alpha-helical structure in aprotic solvent. Steady-state kinetic analysis indicates that the peptide serves as a competitive inhibitor of ERK binding to MEK, with a dissociation constant, Ki, of 0.84 microM. Together with ATP-competitive inhibitors of MEK, we have used this peptide to define the kinetic mechanism of MEK catalysis. These studies reveal that MEK operates through a bi-bi random-ordered sequential mechanism. The synthetic peptide inhibits also the phosphorylation of p38 and ERK by the upstream activator MKK3, but is at least 3-fold less potent as an inhibitor of SEK activation of JNK1. Interestingly, the peptide also showed some ability to inhibit ERK-mediated phosphorylation of myelin basic protein, but was inactive as an inhibitor of the unrelated kinases Raf, Abl, and PKA. These results imply that the alpha C helix is an important locus of interaction for the formation of a MEK-ERK complex. The alpha C helix cannot, however, be the sole determinant of activator selectivity among the MAP kinases. Molecules designed to target the alpha C helix binding pocket of MAP kinase activators may provide a novel means of inhibiting these signal transducers.

Inhibition of mitogen-activated protein kinase kinase blocks T cell proliferation but does not induce or prevent anergy.

Three mitogen-activated protein kinase pathways are up-regulated during the activation of T lymphocytes, the extracellular signal-regulated kinase (ERK), Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase pathways. To examine the effects of blocking the ERK pathway on T cell activation, we used the inhibitor U0126, which has been shown to specifically block mitogen-activated protein kinase/ERK kinase (MEK), the kinase upstream of ERK. This compound inhibited T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs, but had no effect on IL-2-induced proliferation. The block in T cell proliferation was mediated by down-regulating IL-2 mRNA levels. Blocking Ag-induced proliferation by inhibiting MEK did not induce anergy, unlike treatments that block entry into the cell cycle following antigenic stimulation. Surprisingly, induction of anergy in T cells exposed to TCR cross-linking in the absence of costimulation was also not affected by blocking MEK, unlike cyclosporin A treatment that blocks anergy induction. These results suggest that inhibition of MEK prevents T cell proliferation in the short term, but does not cause any long-term effects on either T cell activation or induction of anergy. These findings may help determine the viability of using mitogen-activated protein kinase inhibitors as immune suppressants.

MEK inhibitors: the chemistry and biological activity of U0126, its analogs, and cyclization products.

In search of antiinflammatory drugs with a new mechanism of action, U0126 was found to functionally antagonize AP-1 transcriptional activity via noncompetitive inhibition of the dual specificity kinase MEK with an IC50 of 0.07 microM for MEK 1 and 0.06 microM for MEK 2. U0126 can undergo isomerization and cyclization reactions to form a variety of products, both chemically and in vivo, all of which exhibit less affinity for MEK and lower inhibition of AP-1 activity than parent, U0126.

The p38 mitogen-activated protein kinase pathway in activated and anergic Th1 cells.

Stimulation of T cells through the TCR leads to activation of the mitogen-activated protein kinase (MAPK) family members ERK (extracellular signal-regulated kinase) and JNK (jun NH2-terminal kinase). These kinases act in synergy to increase the activity of the transcription factor AP-1 which is involved in the transcriptional upregulation of IL-2. Recently a third MAPK member, p38, has been identified. The effects of T cell activation on this pathway have not yet been elucidated. Using two murine Th1 clones, we demonstrate that the p38 pathway is induced upon anti-CD3 plus anti-CD28 crosslinking or PMA plus ionomycin stimulation. p38 activity was induced fully by anti-CD3 or PMA alone and is not enhanced by costimulation even at low levels of TCR signaling. p38 activity peaked at 20 min and was significantly decreased by 2 hr. Anergic (tolerant) Th1 cells showed decreased p38 activity as well as decreased ERK and JNK activities even though levels of these proteins remained unchanged.

The effects of IL-1 on mitogen-activated protein kinases in rabbit articular chondrocytes.

IL-1-activated chondrocytes express a large number of genes which contribute to cartilage degradation. The signaling pathways activated in response to IL-1 in these cells are not well-defined. We examined the effects of IL-1 and other stimuli on the mitogen activated protein kinase (MAPK) pathways in rabbit articular chondrocytes. We demonstrate that IL-1 activates three MAPKs, ERK, JNK and p38, in a time and dose-dependent manner. Activation is maximal by 15 minutes and returns to baseline levels by 1 hour. Maximal activation of ERK and p38 occurs with 1 ng/ml IL-1 whereas activation of JNK requires 10-fold higher levels. In contrast to IL-1, the PKC activator, PDBu preferentially activates ERK while TNF alpha preferentially activates JNK. LPS and TGF beta fail to stimulate any of the kinases examined. These results suggest that activation of the various MAPK pathways is important in the response of chondrocytes to IL-1, cytokines and growth factors.

Anergic T cells are defective in both jun NH2-terminal kinase and mitogen-activated protein kinase signaling pathways.

T helper type 1 cells (Th1) become anergic when stimulated through the antigen receptor in the absence of costimulation. They do not produce IL-2 or proliferate in response to subsequent stimulation. Previous studies have indicated that anergic T cells are defective in the trnsactivational activity of the transcription factor, AP-1, which is required for optimal IL-2 transcription. Using two murine Th1 cell clones, we demonstrate that anergic Th1 cells have defects in both jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activities. These kinases have been shown to be important for the upregulation of AP-1 activity. Furthermore, our data show that ERK and JNK activities are restored when anergy is induced in the presence of the protein synthesis inhibitor cycloheximide, or when anergic T cells are allowed to proliferate in response to exogenous IL-2. These treatments have previously been shown to prevent or reverse the anergic state. Our results suggest that defects in both JNK and ERK may result in the decreased AP-1 activity and the reduced IL-2 transcription observed in anergic T cells.

Mice can recover from pulmonary influenza virus infection in the absence of class I-restricted cytotoxic T cells.

Intranasal exposure of athymic (nu/nu) BALB/c mice to influenza virus leads to a persistent infection of the respiratory tract from which the mice die, usually within 3 to 4 wk with symptoms of general cachexia. However, if these nude mice were injected 1 day after infection, with approximately 10(6) cells from individual virus-specific MHC class II-restricted Th cell clones, they showed greatly reduced mortality and the titers of infectious virus in their lungs were reduced, often to undetectable levels. By coinfecting mice with pairs of antigenically distinct viruses and subsequently determining the extent of clearance of each type of virus, it could be shown first that the clearance mechanism was immunologically specific but did not display the typical crossreaction of class I-restricted cytotoxic T (Tc) cells. In addition, neither primary nor memory Tc responses could be detected in these mice. Second, Th cell clones promoted clearance solely of those viruses that contained the specific Th cell determinant, i.e., Th cell-nonreactive bystander viruses were not cleared. These findings were compatible with virus clearance being effected either directly after recognition of infected class II-positive cells by the transferred Th cells or indirectly via promotion of a glycoprotein-specific antibody response. The latter seems to be the case because transfer of Th cells into infected T and B cell-deficient SCID mice did not result in virus clearance, although transfer of an anti-hemagglutinin antibody cocktail did. Thus, a virus-specific Tc cell response is not a requirement for recovery from a pulmonary influenza virus infection.

Characterization of G-protein alpha subunits in the Gq class: expression in murine tissues and in stromal and hematopoietic cell lines.

Murine G alpha 14 and G alpha 15 cDNAs encode distinct alpha subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins). These alpha subunits are related to members of the Gq class and share certain sequence characteristics with G alpha q, G alpha 11, and G alpha 16, such as the absence of a pertussis toxin ADP-ribosylation site. G alpha 11 and G alpha q are ubiquitously expressed among murine tissues but G alpha 14 is predominantly expressed in spleen, lung, kidney, and testis whereas G alpha 15 is primarily restricted to hematopoietic lineages. Among hematopoietic cell lines, G alpha 11 mRNA is found in all cell lines tested, G alpha q is expressed widely but is not found in most T-cell lines, G alpha 15 is predominantly expressed in myeloid and B-cell lineages, and G alpha 14 is expressed in bone marrow adherent (stromal) cells, certain early myeloid cells, and progenitor B cells. Polyclonal antisera produced from synthetic peptides that correspond to two regions of G alpha 15 react with a protein of 42 kDa expressed in B-cell membranes and in Escherichia coli transformed with G alpha 15 cDNA. The expression patterns that were observed in mouse tissues and cell lines indicate that each of the alpha subunits in the Gq class may be involved in pertussis toxin-insensitive signal-transduction pathways that are fundamental to hematopoietic cell differentiation and function.

Activity of CD4+ T-cell clones of type 1 and type 2 in generation of influenza virus-specific cytotoxic responses in vitro.

The activity of distinct CD4+ T-helper cell (Th) clones in promoting secondary A/PR/8/34/Mt.S.(H1N1) (A/PR8) influenza virus-specific, class I-restricted cytotoxic T-lymphocyte (CTL) responses in vitro was examined. CD8+ T cells which had been purified by fluorescence-activated cell sorter from spleen cells of A/PR8-primed mice were used as responders. On their own, purified CD8+ T cells were unable to generate cytotoxic activity upon in vitro culture with A/PR8-infected stimulator cells. Significant cytotoxic activity was generated in cultures that were additionally supplemented with A/PR8-specific Th clones or cell-free supernatant from these clones. Although there were large differences among individual Th clones in this function, Th clones of type 1 (Th1) promoted, on average, significantly stronger cytotoxic responses than Th clones of type 2 (Th2). The differences in promotion of a cytotoxic response correlated with the amount of interleukin-2 (IL-2) or IL-4 secreted by individual Th clones. These two lymphokines accounted for the CTL-promoting activity of the respective Th clones, since addition of recombinant IL-2 (IL-2) or rIL-4 to Th-free cultures substituted fully for the respective Th clones. As observed with Th clones, rIL-2 was significantly more effective than rIL-4 in promoting a cytotoxic response. When used in combination, Th2 clones had an antagonistic effect on the generation of a CTL response by Th1 clones. This effect could be partially transferred with cell-free supernatant from activated Th2 clones and could be reversed by addition of excess rIL-2. Both consumption of IL-2 by Th2 and secretion of an inhibitory factor(s) appear to be involved in this phenomenon.

Identification of eight determinants in the hemagglutinin molecule of influenza virus A/PR/8/34 (H1N1) which are recognized by class II-restricted T cells from BALB/c mice.

Eight nonoverlapping regions of the hemagglutinin (HA) molecule of influenza virus A/PR/8/34 (PR8), which serve as recognition sites for class II-restricted T cells (TH) from BALB/c mice, have been identified in the form of 10- to 15-amino-acid-long synthetic peptides. These TH determinants are located between residues 110 to 313 of the HA1 polypeptide. From a total of 36 HA-specific TH clones and limiting-dilution cultures of independent clonal origins, 33 (90%) responded to stimulation with one of these peptides. The residual three TH clones appeared to recognize a single additional determinant on the HA1 polypeptide which could not be isolated, however, in the form of a stimulatory peptide. None of the motifs that have been proposed to typify TH determinants were displayed by more than half of these recognition sites. Most unexpected was the finding that none of the TH determinants was located in the ectodomain of the HA2 polypeptide that makes up roughly one-third of the HA molecule. Possible reasons for the preferential recognition of HA1 as opposed to HA2 by TH are discussed.

A new mouse T-cell receptor alpha chain variable region family.

Sequence analysis of the rearranged T-cell receptor alpha chain gene segments from an influenza reactive T-cell clone T2.5-5 and a hemin chloride reactive T-cell hybrid SJL-HE-1.1 have revealed a previously undescribed V alpha gene family. We have designated this family V alpha 15. Southern hybridization analysis has indicated that this family most probably contains only two members, and that these are conserved in each of six mouse strains representing three previously described V alpha haplotypes: V alpha a, V alpha b, and V alpha c.