Regulation of T-lymphocyte mitogenesis by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE)

Synthesis of lipoxygenase metabolites of [¹⁴C]arachidonic acid by mouse spleen lymphocyte cultures was inhibited by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE) in a dose-dependent manner. In parallel experiments, the influence of 15-HETE on mitogenesis in spleen lymphocyte cultures was examined. 15-HETE at concentrations similar to those which inhibited cellular lipoxygenases progressively inhibited mitogenesis induced by the T-cell mitogen PHA but had no significant effect on the mitogenic response to the B-cell mitogen LPS. The inhibitory response was maximal when 15-HETE was added within 8 hr of exposure to PHA. Several analogs of 15-HETE having progressively fewer double bonds were tested in the same systems. 15-OH,20:3 had approximately the same potency as 15-HETE in inhibiting both mitogenesis and formation of metabolites from [¹⁴C]arachidonic acid. 15-OH, 20:2 and 15-OH,20:0 were much less active in either assay. Mitogenesis, induced in spleen cell cultures by the tumor promoter phorbol myristate acetate, was also blocked by 15-HETE. These experiments indicate that lipoxygenase metabolites of arachidonic acid may play an important role in T-lymphocyte blastogenesis and suggest that 15-HETE, via its ability to selectively inhibit cellular lipoxygenases, may function as an endogenous regulator of T-lymphocyte responses.     

CD40 ligand-deficient mice generate a normal primary cytotoxic T-lymphocyte response but a defective humoral response to a viral infection.

CD40 ligand is expressed on activated T cells and interacts with CD40 on B cells and monocytes. It is not known what role CD40 ligand plays in the generation of immune responses to viral infection. To address this issue, we examined virus-specific T- and B-cell responses in CD40 ligand-deficient (CD40L-/-) mice following infection with lymphocytic choriomeningitis virus (LCMV). We found that primary anti-LCMV specific antibody responses were severely impaired in CD40L-/- mice, with the defect being most striking for antibody of the immunoglobulin G1 (IgG1) isotype. Interestingly, low levels of LCMV-specific antibodies of the IgG2a, IgG2b, and IgG3 isotypes were made in the CD40L-/- mice, showing that IgG1 responses are totally dependent on CD40L but that at least some IgG2a, IgG2b, and IgG3 responses can be CD40L independent. However, unlike CD40L+/+ mice, CD40L-/- mice were unable to sustain virus-specific antibody responses and showed a gradual decline in serum antibody levels over time. The CD40L-/- mice were also deficient in the generation of memory B cells. In contrast to the severely impaired humoral responses, CD40L-/- mice generated potent virus-specific CD8+ cytotoxic T-lymphocyte responses after LCMV infection and were able to clear the virus. These results show that CD40L does not play a role in generating primary virus-specific CD8+ cytotoxic T-lymphocyte responses but does affect the primary antibody response and the generation of memory B cells.     

ACTH receptor distribution and modulation among murine mononuclear leukocyte populations

Murine mononuclear leukocytes express adrenocorticotropin (ACTH) receptors that were recognized by a monospecific antiserum to the ACTH receptor on Y-1 adrenal cells. The antiserum was utilized in an immunofluorescence (IF) assay to characterize the distribution of ACTH receptors on resting murine mononuclear leukocyte populations. Forty-seven percent of spleen cells, 32% of lymph node cells, and 1% of thymocytes constitutively expressed ACTH receptors. Separation of lymphocytes into purified B cell and T cell populations, followed by IF analysis revealed that 47% of B cells and 23% of T cells possessed ACTH receptors. Helper T cells (CD4+ T cells) constituted the majority of ACTH receptor-positive T lymphocytes. Furthermore, 47% of resident peritoneal macrophages, purified by adherence to plastic, expressed ACTH receptors. The T-lymphocyte mitogen, concanavalin A, interferon gamma, and ACTH enhanced ACTH receptor expression. The differential distribution of ACTH receptor-positive cells among specific leukocyte populations explains in part why differential cellular responses are observed and implies important regulatory functions for these receptors in the generation or regulation of immune responses.     

Inhibition of T-lymphocyte mitogenic responses and effects on cell functions by bovine herpesvirus 1.

The mitogenic response of bovine peripheral blood mononuclear cells stimulated by concanavalin A (ConA) was suppressed by infectious bovine herpesvirus 1 (BHV-1). Proliferation in response to interleukin-2 (IL-2) by IL-2-dependent lymphocyte cultures was also inhibited by BHV-1. Although inhibition of mitogenesis approached 100%, less than 1 cell in 1,000 was productively infected by BHV-1 in ConA-stimulated cultures. Neither conditioned medium from mitogen-stimulated peripheral blood mononuclear cell cultures nor human recombinant IL-2 reversed suppression by the virus. Infection by BHV-1 did not influence the expression of IL-2 or IL-2 receptor mRNA in ConA-stimulated cultures, nor did it affect the cytolytic capabilities of lymphocytes. The data suggest that the inhibition of T-lymphocyte proliferation is the result of a nonproductive BHV-1 infection.     

Contrasting effects of T cell growth factors on T cell responses to melanoma in vitro

 Determinants of T cell responses to tumor cells remain largely unknown. In the present study we have used long-term cultures of human melanoma cells and autologous peripheral blood lymphocytes to examine the influence of cytokines with T cell growth activity on the phenotype and cytotoxic and proliferative response of T cells to melanoma. It was found that addition of interleukin-4 (IL-4) inhibited the response of CD8⁺ T cells and promoted the response of the CD4 subset. IL-2 or IL-7 was effective in increasing melanoma-specific cytotoxic T lymphocyte (CTL) activity in cultures where CD8 T cells were predominant, whereas IL-4 followed by IL-2 was most effective in cultures where CD4 T cells predominated. IL-10 or IL-12 inhibited proliferation and CTL activity against melanoma in long-term cultures. The effects of IL-12 were reproduced in long-term cultures of T cells stimulated with mAb against CD3 and were shown to depend on prior exposure of T cells to IL-12 before IL-2. As yet unidentified factors, such as co-factor expression on melanoma, appear to be as important as exogenous cytokines in determining the nature of T cell responses to melanoma. These results suggest that analysis of responses in long-term culture may assist in defining the role of key cytokines and other determinants of immune responses to melanoma. Received: 4 June 1996 / Accepted: 12 November 1996     

Immunomodulation by cocaine--a neuroendocrine mediated response

Cocaine acts directly on lymphoid cells and indirectly modulates the immune response by affecting the level of neuroendocrine hormones. In vitro, very high concentrations of cocaine inhibit different immune responses, while plasma levels following cocaine use have no effect. The results of the few published in vivo studies are contradictory, showing stimulatory, suppressive or no effect on lymphoid cells. The indirect effects of cocaine on the immune system could be mediated by ACTH, beta-endorphin and corticosterone. Anorectic effect associated with nutritional deficiencies of drug users could additionally affect the immune response by cocaine.     

Liposomes in Host Defense Mechanism: A Key Role for Macrophages

Abstract

Liposomes can be used as carriers for antigens, immunomodulators and cytotoxic drugs. Such liposomes may serve as a tool to manipulate immune and non-immune host defense mechanisms. In most cases their effects are mediated by macrophages. Macrophages seem to be involved in humoral (antibody) responses and in cytotoxic T-lymphocyte responses. They are also important in non-immune defense mechanisms against foreign invaders and altered self. Which macrophages can be influenced by the liposome encapsulated molecules depends on the administration route of the liposomes. The macrophages ingest the liposomes. Once within the cell, lysosomal phospholipases disrupt the phospholipid bilayers. In this way, encapsulated molecules are released in the cell. Such liposome delivered molecules can be processed (antigens), activate the macrophage (immunomodulators) or disturb the metabolism of the cells (cytotoxic drugs). That the latter inhibition of macrophage functions may result in immunopotentiation is explained by the fact that certain macrophages are regulating immune functions by suppression.     

On the mechanisms of inhibition of lymphocyte mitogenesis by high conA concentrations

ConA at high concentrations inhibits lymphocyte mitogenesis. Previous studies have shown that inhibitory conA concentrations do not inhibit the acquisition of responsiveness to interleukin-2 (IL-2) when excessive conA is removed. To analyse further the problem of high-dose inhibition by conA, we determined whether inhibition of mitogenesis is related to inhibition of IL-2 production or, alternatively, whether factor production is intact, but the cells are rendered incapable of responding to the factor. ConA stimulates IL2 production at concentrations that are inhibitory to mitogenesis of human lymphocytes. IL-2 was assayed both in a murine cytotoxic T cell line and human memory cells. The response of IL-2-dependent cells to IL-2-containing medium was, on the other hand, inhibited by conA in a dose-dependent fashion. One mechanism whereby high conA concentrations inhibit mitogenesis is by rendering cells resistant to IL-2, possibly via extensive cross-linking of cell surface sites.     

Inhibition of proliferation without affecting the generation of cytotoxicity in the human mixed lymphocyte reaction

Phosphoramide mustard (PM) is considered to be the major tumoricidal metabolite of cyclophosphamide in vivo. The effects of this metabolite in vitro on several immune functions of human lymphocytes have been investigated. Very low concentrations (10(-7) to 10(-9) M) of PM added to lymphocyte cultures inhibited proliferation of the lymphocytes in response to mitogens and alloantigens. At these concentrations, inhibition of proliferation appeared to be due to a direct action of PM on the proliferative cells. Thus, concanavalin A-stimulated lymphocytes still acquired IL-2 receptors (Tac antigen) normally in the presence of PM (10(-6) to 10(-9) M). Only exceedingly high concentrations of PM (10(-5) M or greater) prevented the acquisition of Tac antigen. Similarly, the inhibition of proliferation was probably not related to endogenous IL-2 levels: addition of exogenous IL-2 to PM-containing cultures did not result in any restoration of proliferation. Further evidence that PM directly affected proliferative cells was that low concentrations of PM inhibited the proliferation of T cells continuously growing in IL-2. The exposure time to PM necessary for inhibition was essentially identical to those for lymphoproliferative responses to mitogens and alloantigens. Paradoxically, however, the generation of cytotoxic lymphocytes in mixed lymphocyte reactions (MLRs) and mixed lymphocyte tumor cell cultures (MLTCs) was very resistant to PM. In parallel MLRs and MLTCs the cytotoxic responses were resistant to approximately 1000-fold more PM than were the proliferative responses. Only at 10(-5) M PM were these inhibited. These data suggest that clonal expansion of cytotoxic lymphocytes or their precursors by proliferation is not an absolute requirement for the generation of cytolytic activity.     

Inhibition of T cell responses to alloantigens and polyclonal mitogens by Ly-5 antisera

The effects of Ly-5 alloantisera on the generation of cytotoxic T cells (CTL), on the effector phase of CTL killing, and on polyclonal mitogenesis were studied. Ly-5 antisera added at the beginning of mixed lymphocyte culture (MLC) suppressed the production of CTL in an allele-specific manner. Neither Ly-5.1 nor Ly-5.2 antisera inhibited the generation of cytotoxic effectors by Ly-5.1/Ly-5.2 heterozygous spleen cells; however, a combination of Ly-5.1 and Ly-5.2 antisera markedly suppressed the appearance of Ly-5 heterozygous CTL. Similarly, Ly-5 antisera inhibited the effector phase of CTL killing in an allele-specific manner. In addition, Ly-5 alloantisera specifically blocked concanavalin A and oxidative mitogenesis of splenocytes carrying the appropriate Ly-5 alloantigen. The results are discussed in light of a possible functional role of Ly-5 molecules in immune processes.     

Regulation of lymphokine (gamma-interferon) production by corticotropin

We have shown that corticotropin (ACTH), alpha-endorphin, and enkephalins can regulate antibody responses, which suggested a role for neuropeptides in a regulatory circuit between the immune and neuroendocrine systems. ACTH and structurally related peptides were examined here for regulation of mitogen induction of the lymphokine gamma-interferon (IFN gamma) in C57BL/6 mouse spleen cell cultures. Synthetic ACTH1-39 and a porcine pituitary extract containing ACTH activity were potent suppressors of the IFN gamma response. Synthetic ACTH1-39 suppressed the response by approximately 62% at 1 to 3 microM, whereas the porcine extract suppressed by greater than 90% at 1 to 3 microM ACTH. The greater potency of the pituitary extract was shown to be due to the presence of an additional peptide of Mr 2100 that was reactive with antibodies to the N-terminal region of ACTH (ACTH1-13), possessed potent anti-cellular activity against L cells and various transformed cells, but lacked ACTH biologic activity. The anti-cellular peptide suppressed the IFN gamma response by greater than 99% at 0.05 microM. The ACTH1-39 cleavage products, alpha-melanocyte stimulating hormone (alpha MSH; acetylated and amidated ACTH1-13), and corticotropin-like intermediate lobe peptide (CLIP; ACTH18-39) had no effect on IFN gamma production. ACTH1-24, like ACTH1-39, has full steroidogenesis activity but also had no effect on IFN gamma production, which suggests a dissociation of the immunoregulatory and steroidogenic properties of ACTH1-39. ACTH1-39, and possibly also the anti-cellular 2100 Mr peptide, is initially synthesized as the precursor polyprotein pro-opiomelanocortin (POMC). Enzymatic processing of POMC, first to the active ACTH1-39 or the anti-cellular peptide and then to the inactive smaller peptides, probably plays an important role in regulation of lymphokine and antibody production by ACTH and ACTH-related neuropeptides. This is consistent with the recent demonstration of the production of ACTH-like peptides by lymphocytes.     

Human Memory Cytotoxic T-Lymphocyte (CTL) Responses to Hantaan Virus Infection: Identification of Virus-Specific and Cross-Reactive CD8+ CTL Epitopes on Nucleocapsid Protein

Hantaan virus, the prototypic member of the Hantavirus genus, causes hemorrhagic fever with renal syndrome in humans. We examined the human memory T-lymphocyte responses of three donors who had previous laboratory-acquired infections with Hantaan virus. We demonstrated virus-specific responses in bulk cultures of peripheral blood mononuclear cells (PBMC) from all donors. Bulk T-cell responses were directed against either Hantaan virus nucleocapsid (N) or G1 protein, and these responses varied between donors. We established both CD4(+) and CD8(+) N-specific cell lines from two donors and CD4(+) G1-specific cell lines from a third donor. All CD8(+) cytotoxic T-lymphocyte (CTL) lines recognized one of two epitopes on the nucleocapsid protein: one epitope spanning amino acids 12 to 20 and the other spanning amino acids 421 to 429. The CTL lines specific for amino acids 12 to 20 were restricted by HLA B51, and those specific for amino acids 421 to 429 were restricted by HLA A1. The N-specific CTL lines isolated from these two donors included both Hantaan virus-specific CTLs and hantavirus cross-reactive CTLs. Responses to both epitopes are detectable in short-term bulk cultures of PBMC from one donor, and precursor frequency analysis confirms that CTLs specific for these epitopes are present at relatively high precursor frequencies in the peripheral T-cell pool. These data suggest that infection with Hantaan virus results in the generation of CTL to limited epitopes on the nucleocapsid protein and that infection also results in the generation of cross-reactive T-cell responses to distantly related hantaviruses which cause the distinct hantavirus pulmonary syndrome. This is the first demonstration of human T-lymphocyte responses to Hantaan virus.     

Nonmyeloablative immunosuppressive regimen prolongs In vivo persistence of gene-modified autologous T cells in a nonhuman primate model

The in vivo persistence of gene-modified cells can be limited by host immune responses to transgene-encoded proteins. In this study we evaluated in a nonhuman primate model whether the administration of a nonmyeloablative regimen consisting of low-dose total-body irradiation with 200 cGy followed by immunosuppression with mycophenolate mofetil and cyclosporin A for 28 and 35 days, respectively, could be used to facilitate persistence of autologous gene-modified T cells when a transgene-specific immune response had already been established or to induce long-lasting tolerance in unprimed recipients. Two macaques (Macaca nemestrina) received infusions of T cells transduced to express either the enhanced green fluorescent protein and neomycin phosphotransferase genes or the hygromycin phosphotransferase and herpes simplex virus thymidine kinase genes. In the absence of immunosuppression, both macaques developed potent class I major histocompatibility complex-restricted CD8(+) cytotoxic T-lymphocyte (CTL) responses that rapidly eliminated the gene-modified T cells and that persisted long term as memory CTL. Treatment with the nonmyeloablative regimen failed to abrogate preexisting memory CTL responses but interfered with the induction of transgene-specific CTL and facilitated in vivo persistence of gene-modified cells in an unprimed host. However, sustained tolerance to gene-modified T cells was not achieved with this regimen, indicating that further modifications will be required to permit sustained persistence of gene-modified T cells.     

Generation and characterization of B7-H4/B7S1/B7x-deficient mice

Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.     

Trophoblast regulation of maternal-paternal lymphocyte interactions

The modification of immunological responses by murine embryonic trophoblast cells was investigated using the mixed-lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) test. In MLR containing Balb/c (responder) and C57BL/6 (stimulator) splenocytes DNA synthesis was markedly reduced in the presence of ectoplacental cone or placental trophoblast cells. These same trophoblast cell populations inhibited the in vitro generation of cytotoxic lymphocytes while cultures containing nontrophoblast regulator cells expressed normal cytotoxicity. DNA synthesis in MLR and cytotoxic activity in CML were not suppressed in cultures containing $\text{3}\text{1}\text{2}$ day blastocyst outgrowths. The potent immunosuppressive properties of the trophoblast may be important during pregnancy by protecting the genetically dissimilar fetus from potentially harmful maternal immune effector mechanisms.     

In vitro generation of secondary cell-mediated cytotoxic response against a syngeneic Gross virus-induced lymphoma in rats.

Cell-mediated cytotoxic responses against a syngeneic Gross virus-induced lymphoma, (C58NT)D, in W/Fu rats were generated in vitro by using mixed lymphocyte-tumor cell cultures. The source of responding cells was either spleens from normal rats or spleens from rats carrying or having rejected (C58NT)D tumors. Mitomycin C-treated (C58NT)D tumor cells were used as stimulating cells. The secondary anti-tumor cytotoxic response occurred more rapidly and reached higher levels than the primary response, and it was antigen specific. T cells, but nor B cells or macrophages, were essential for both the induction and the effector phases of the secondary anti-tumor responses. These data suggest that specific memory T cells persist for long periods of time in the lymphoid organs of (C58NT)D immune rats, which can rapidly become cytotoxic upon re-exposure to antigen.     

CD4+ T-cell-epitope escape mutant virus selected in vivo.

Mutations in viral genomes that affect T-cell-receptor recognition by CD8+ cytotoxic T lymphocytes have been shown to allow viral evasion from immune surveillance during persistent viral infections. Although CD4+ T-helper cells are crucially involved in the maintenance of effective cytotoxic T-lymphocyte and neutralizing-antibody responses, their role in viral clearance and therefore in imposing similar selective pressures on the virus is unclear. We show here that transgenic virus-specific CD4+ Tcells, transferred into mice persistently infected with lymphocytic choriomeningitis virus, select for T-helper epitope mutant viruses that are not recognized. Together with the observed antigenic variation of the same T-helper epitope during polyclonal CD4+ T-cell responses in infected pore-forming protein-deficient C57BL/6 mice, this finding indicates that viral escape from CD4+ T lymphocytes is a possible mechanism of virus persistence.     

Ascorbic acid accumulation by cultured rat adrenocortical cells

Summary The influence of adrenocorticotrophin (ACTH) on radiolabeled ascorbic acid (AA) accumulation by adrenocortical cells was examined in primary cultures of collagenase dissociated glands from adult male rats. The cells were ACTH responsive by morphological and steroidogenic criteria. After 5 d in AA-free medium, cells pretreated with 100 mU/ml ACTH for 3 d took up two to three times more AA over a 2 h period than did untreated controls (4.0 to 10.0 nmol versus 1.7 to 3.4 nmol AA/μg DNA). In contrast, ACTH administered on Day 6 concurrently with AA inhibited AA accumulation compared to cultures exposed to AA alone. This acute inhibitory effect of ACTH was in the order of 30% in cultures pretreated with ACTH for 3 d but was not significant (7%) without ACTH pretreatment. The results show that ACTH has distinct long term stimulatory and acute inhibitory effects on AA accumulation by adrenocortical cells and suggest that both maximal AA accumulation and the responsiveness to acute inhibition of AA accumulation by ACTH may depend on the maintenance of the differentiated state of the adrenal cortex. This work was supported by a grant and research associateship to N. A. from the National Cancer Institute of Canada.     

Analysis of T-cell subset proliferation at afebrile and febrile temperatures: differential response of Lyt-1+23- lymphocytes to hyperthermia following mitogen and antigen stimulation and its functional consequence on development of cytotoxic lymphocytes

Poikilotherms are now known to increase their survival by behaviorally induced fevers in response to pathogenic infection. Increased host resistance to viral and bacterial infections has also been noted in homeotherms whose body temperature has been elevated by manipulation of ambient temperature. These observations suggest that fever may increase host resistance by augmenting acquired immunity; thus, this highly conserved response during evolution may provide a survival advantage against environmental pathogens. This possibility has prompted us to investigate the influence of a temperature characteristic of a modest fever in humans (39 degrees C) on T-cell proliferation and function. Our studies revealed that T-cell mitogenesis was enhanced when cultures were incubated at the febrile temperature (39 degrees C). Analysis of T-cell subsets demonstrated that temperature enhanced the mitogenic (Concanavalin A) response of Lyt-1+23- splenocytes; in contrast, hyperthermia was deleterious to lectin-driven proliferation of the Lyt-1-23+ population even in the presence of large quantities of recombinant interleukin-2 (rIL-2). B-cell mitogenesis was invariably inhibited by hyperthermia over a broad range of concentrations of lipopolysaccharide (LPS). Although T-cell mitogenesis was enhanced at the febrile temperature, T-cell proliferation induced by alloantigens or by a murine pathogen, Sendai virus (SV), was diminished at the febrile temperature. Hyperthermia inhibited SV-induced proliferation of Lyt-1+23- lymphocytes, indicating that a febrile temperature can either augment or inhibit T-cell proliferation of the same T-cell subset depending upon the activation signal (i.e., lectin or antigen). Because effector cell development depends upon antigen-induced clonal expansion (proliferation), we evaluated the influence of temperature on primary cytotoxic thymus (T)-derived lymphocyte (CTL) responses against alloantigens and secondary CTL responses against SV under afebrile and febrile conditions. We consistently observed that the induction of alloreactive and virus-specific CTL was diminished in cultures incubated at the elevated temperature, suggesting that a thermosensitive event(s) exists in the progression of CTL derived from either CTL precursors (CTLp) or memory CTL. Furthermore, hyperthermia reduced the number of SV-specific CTL detectable by limiting dilution analysis, suggesting that another event independent of clonal expansion was thermolabile during effector cell development. In view of these results, we suggest that it may be premature to conclude that the observed increase in host resistance induced by a febrile state is mediated by enhanced cell-me