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The in vivo effects of BCG were studied on antibody-dependent cellular cytotoxicity (ADCC) using an assay in which the effectors were spleen cells of BCG-treated mice, and the targets antibody-coated chicken red blood cells (CRBC). In the study of the time course of action, an increased ADCC-activity was observed starting at day 10 with a maximum (four-fold augmentation) at day 14 after BCG treatment. The nature of the effector cell was investigated: in normal mice the activity was found in two cell populations, i.e. macrophages and theta-negative (θ-) nylon-adherent spleen cells. A BCG treatment increased the ADCC activity of macrophages of θ- nylon-adherent spleen cells and induced a population of θ- nylon-non-adherent spleen cells. In old mice, the effects of BCG were compared after a weekly long-term (6 months) treatment and after administration of a single dose. It was concluded that ADCC is higher in old mice than in young mice and can be further increased by treatment with a single dose of BCG in contrast with the long-term treatment which impaired this immune reaction. [ABSTRACT FROM AUTHOR]

An attempt to correct the state of immunodeficiency in old age was made by repeatedly injecting a chemically defined immunostimulating agent, bestatin, to 16 month old (C57B1/6 × BALB/c) F1 mice. Aged mice were found to have depressed T-cell and B-cell responses but increased ADCC activity. Weekly injections of bestatin over a period of 6 months resulted in varying effects depending on the dose administered. Small doses (10 μg per injection) were more effective m restoring humoral responses to SRBC rather than delayed-type hypersensitivity reactions, whereas large doses (100 μg per injection) acted in the opposite way. Macrophage activation was only obtained after the administration of the high doses of bestatin. Continuous treatment with bestatin did not prevent the appearance of suppressor cells induced by ageing. It led to a significant reduction of ADCC activity in aged animals near to the base line value of young animals. Animals were examined for the presence of spontaneous tumours from the end of the treatment until the age of 28 months. A significant reduction of spontaneous turnout incidence was observed in mice given repeated injections of 100 μg bestatin when compared to untreated aged mice and to mice given the low doses of bestatin. [ABSTRACT FROM AUTHOR]

Direct RNA nanopore sequencing reveals changes in gene expression, polyadenylation, splicing, m6A methylation, and pseudouridylation in response to influenza virus exposure in primary human bronchial epithelial cells. This study focuses on the epitranscriptomic profile of genes in the host immune response. In addition to polyadenylated noncoding RNA, we purified and sequenced nonpolyadenylated noncoding RNA and observed changes in expression, N6-methyl-adenosine (m6A), and pseudouridylation (Ψ) in these novel RNA. Two recently discovered lincRNA with roles in immune response, Chaserr and LEADR , became highly methylated in response to influenza exposure. Several H/ACA type snoRNAs that guide pseudouridylation are decreased in expression in response to influenza, and there is a corresponding decrease in the pseudouridylation of two novel lncRNA. Thus, novel epitranscriptomic changes revealed by direct RNA sequencing with nanopore technology provides unique insights into the host epitranscriptomic changes in epithelial gene networks that respond to influenza virus infection.

The Fas/Fas ligand (FasL) pathway is one of the two major effector mechanisms of T cell-mediated cytotoxicity. To prevent nonspecific killing by lymphoid cells, FasL expression on the cell surface of immune effector cells is strictly regulated. However, MRL/lpr autoimmune-prone mice massively overexpress FasL on their T lymphocytes, which render them able to kill Fas+ targets in vitro and in vivo. It is surprising that we show in the present work that B lymphocytes purified from MRL/lpr spleen cells express FasL to the same extent as T cells at the mRNA and protein level. These B cells are potent cytotoxic effectors against Fas+ but not Fas- targets. The B lymphocyte effectors were used ex vivo without any in vitro activation by B cell stimuli. Furthermore, we found that MRL/lpr B lymphocytes have the same cytotoxic potential as natural killer cells, which have been characterized as potent, Fas-mediated, cytotoxic effectors. The level of membrane-anchored FasL increases with the size of the B cell and cell-surface activation marker CD69 expression, indicating that the expression of FasL is up-regulated in parallel with the activation state of the B cell. The activated B cell population contained the major cytotoxic activity, and a minor part was associated with CD138/Syndecan-1+ plasma cells.

LINE-1 are endogenous mobile genetic elements that have dispersed and accumulated in the genomes of eukaryotes via germline transposition, with up to 100,000 copies in mammalian genomes. LINE-1 elements transpose by reverse transcription of their own transcript. Transposition requires synthesis of a full-length, sense-strand transcripts and proteins encoded by open reading frame (ORF) 1 and ORF2. Although severely repressed in most normal tissues, LINE-1 occasionally leads to disease by insertional mutagenesis. In the present study, Northern blot and in situ hybridization analyses revealed a template-strand transcription of LINE-1 ORF2 (encoding reverse transcriptase, RT) in lymphoid organs and the liver from MRL-+/+ and Fas-deficient MRL/lpr strains and their normal ancestors. While these sense transcripts are restricted to the nucleus in hepatocytes, they are also found in the cytoplasm in splenocytes. In contrast to transcription, ORF2 translation was detected only in MRL strains, as shown by the cytoplasmic labelling of splenic cells obtained with a monoclonal antibody recognizing the LINE-1 RT. This antibody coprecipitated two proteins of 45 and 12 kDa from MRL/lpr lymphoid organ lysates that were removed by pretreatment with anti-beta2-microglobulin antiserum, suggesting a structural association between a LINE-1 product and a major histocompatibility complex class I or class I-like molecule.

Autoimmune-prone MRL/lpr mice, homozygous for the lpr mutation, exhibit defective apoptosis and develop generalized lymphoproliferation with the accumulation of a double-negative (DN: CD4- CD8-) T cell population. The capacity of lpr T lymphocytes to effectuate Fas- and perforin-mediated cytotoxicity was investigated. Spleen and lymph nodes cells spontaneously lyse Fas- targets (thymocytes) through a Fas-mediated mechanism as a consequence of their overexpression of Fas ligand (FasL) confirmed by semiquantitative reverse transcription (RT)-PCR and immunoprecipitation analysis. This cytotoxicity was greatly increased after stimulation of the effectors by phorbol myristate acetate (PMA) + ionomycin. Under these conditions, MRL/lpr spleen and LN cells exhibited strong Fas-mediated Ca2+-independent cytotoxic activity against wild-type Fas+ (H-2 compatible or incompatible) thymocytes or lipopolysaccharide (LPS)-transformed blast cells. Such Fas-mediated cytotoxic activity was also observed with C57BL/6-lpr, but never with wild-type C57BL/6 or MLR+/+ effectors. Depletion experiments showed that the effector cells of this Fas-mediated cytotoxicity were DN T cells. This subset, which represent in vivo activated T cells, can spontaneously lyse Fas+ targets by a mechanism that does not need the interaction of the T cell receptor (TCR) with major histocompatibility complex molecule plus antigen. This lytic potential is increased by PMA + ionomycin, which sends a second activation signal to these primed T cells. Therefore, the small amounts of Fas receptor expressed on MRL/lpr tissues may account for their nonspecific autoimmune attack by DN cells. In Con A-containing medium, which allows detection of the perforin-mediated pathway against Fas targets, cytotoxic CD8+ effectors were detected that are able to kill lpr thymocytes via a Ca2+-dependent pathway. Thus, in MRL/lpr mice, these CD8+ cells could constitute potent cytotoxic effectors against cells presenting antigen to their TCR.

Cell lines and a clone established from the C57BL/6 (H-2b) Lewis lung (3LL) tumour were previously characterized with respect to tumour growth and metastatic spread in vivo, and to the expression of a 3LL tumour-specific antigen (3LL TA) using a monoclonal antibody raised in syngeneic mice immunized with 3LL cells. No correlation was observed between the presence of 3LL TA and the prevention of metastatic spread which suggests that the immune recognition of this tumour antigen requires the presence of a self H-2 molecule absent from these tumour cells. Indeed, radioimmunoassay (RIA) and cytofluorometric analysis using specific monoclonal antibodies have shown that the H-2Kb molecule was not expressed at the cell surface of all 3LL cell lines and clones, while the H-2Db molecule was present at normal levels. This defect, which was not the consequence of a lack of beta 2m expression, was accompanied by an absence or a marked reduction of the H-2K mRNA level (which has been reversed in the M4 cell line by in vitro gamma interferon treatment), while the H-2D class I gene was normally transcribed. Another defective transcription was also observed for a gene in the Tla region (gene 37). This low '37' phenotype was corrected by in vitro treatment of the M4 cell line with gamma interferon, which indicates that this class I gene of the Qa/Tla region has an interferon response sequence in the promoter.

Cloned malignant cell lines from primary tumor sites (two lines) and from a lung metastatic focus (one line) were established 14 and 28 days after s.c. inoculation of Lewis lung tumor cells into C57BL/6 mice. Cloning was done in semisolid agarose culture medium and individual clones expanded in liquid medium. In vivo malignancy of the three lines was assured by graft experiments to healthy C57BL/6 recipients. Morphology, cloning efficiency in agarose, and growth rate were the same for all three lines. However, the cloned line from a metastatic focus had a higher metastatic rate (number of lung metastases per 10(5) injected tumor cells) compared to the two other cell lines. T-lymphocyte-depleted mononuclear spleen cells from mice grafted with tumor cells 2 to 4 weeks previously were found to be cytotoxic to cells from all three lines, whereas unfractionated mononuclear spleen cells from the same animals had weak or no cytotoxic activity. The cytotoxicity of T-lymphocyte-depleted spleen cells was found to include other malignant and nonmalignant target cells of C57BL/6 origin, but not allogeneic cells. In mixing experiments, splenic T-lymphocytes inhibited the cytotoxic activity of non-T-lymphocytes. The high- and low-metastatic tumor cell lines were found to be equally sensitive to lysis by T-cell-depleted spleen cells, suggesting that the effector cells (cytotoxic autoreactive cells) may have a significant antineoplastic potential.

The MRL/lpr (H-2k) inbred strain, a model for the autoimmune disease systemic lupus erythematosus, differs from the healthy inbred strain MRL +/+ (H-2k) by only 0.1% of its genome. Southern blot analysis using class I and class II probes confirmed the H-2k genotype of both strains. Among the Iak-positive peritoneal cells, cells with an unexpected expression of Iad specificities were detected in a radioimmunoassay using several monoclonal antibodies and one conventional antiserum. This was only found in aged (6- to 9-month-old) mice both in the MRL/lpr strain (32% Iad-positive mice) and in the MRL +/+ strain (42% Iad-positive mice). Furthermore, 24% of aged MRL/lpr mice exhibited strong spontaneous cytotoxic T lymphocyte (CTL) activities against P815 (H-2d) target cells, and 57% had a weaker but still detectable level of cytotoxicity. In contrast, such a CTL activity has never been found in the MRL +/+ strain. These results suggest that the anti-H-2d CTL plays a role in the onset of the autoimmune process in MRL/lpr mice.

A thymic extract which was previously demonstrated to contain a lymphocyte-inhibiting-factor (LIFT) based on its immunosuppressive activity in vivo and its antiproliferative properties on lymphocytes in vitro, has been purified using ultrafiltration procedures. Most of the activity measured by the ability to inhibit DNA synthesis in short term cultures of mouse thymocytes, was recovered in the 10,000-50,000 m. wt fraction (I fraction). In contrast, similar extracts from non-lymphoid organs were always ineffective in decreasing DNA synthesis in thymocytes. The I fraction was also inhibitory of DNA synthesis in mouse spleen cells stimulated by PHA whatever the time at which the fraction was added after the mitogen stimulation. This fraction, as well as the crude thymic extract, was ineffective in decreasing DNA synthesis in non-lymphoid target cells. The I fraction was further purified by Sephadex G50 filtration and DEAE Sephadex chromatography. The active molecule seemed to be a heat resistant basic peptide probably bound to a ribonucleotide moiety.

CBA/J (H-2k) females, mated with DBA/2 J (H-2d) or DBA/1 J (H-2q) males, exhibit a high rate of fetal resorption. In contrast, when H-2 identical CBA substrains (i.e. CBA/Ca and CBA/N) are used, this phenomenon is not observed. On the other hand, before mating with DBA/2 J males, pre-immunization of CBA/J females with spleen cells coming from BALB/c J or (DBA/2 x BALB/c J) F1 males (and not from other H-2d identical males whatever their Mls alleles) has significantly decreased the fetal resorption rate. Thus, immunization against determinants other than classical H-2d (K, I, D, L) antigens (transmitted as a dominant character and different from Mls determinants) can elicit anti-abortive effects. Furthermore, it was observed that the spleen cell endowed with the anti-abortive effects was neither a T nor a B lymphocyte. In contrast, peritoneal cells were able to reproduce the phenomenon, indicating that it may be mediated by a cell of the macrophage-monocyte lineage. Finally, a first gestation was substituted for allo-immunization of CBA/J females. The anti-abortive effects of a first pregnancy by BALB/c J males (and not by other H-2k syngeneic or H-2d allogeneic males) was observed in the course of a second pregnancy sired by DBA/2 J males. These data can be interpreted in terms of maternal recognition of an antigen present on both macrophages and trophoblast cells and necessary for a successful gestation, which is coded for by genes outside the K, I, D, L regions.

Because of the experimental and clinical studies which have been extensively conducted with bacillus Calmette-Guérin (BCG) as a systemic adjuvant in cancer immunotherapy, we have analyzed the main factors and conditions which determine its beneficial action and have underlined some of these (eg, the dose factor which controls the amplification of suppressor cells which is probably responsible for failures and even the possible tumor-enhancing effect of immunotherapy). Knowing those factors and conditions, we have been able to establish a systematic immunopharmacologic study of systemic immunity adjuvants, which has resulted in the discovery of agents whose actions are more rapid than that of BCG on one or a few populations of cells involved in immunity and which, unlike BCG, do not induce suppressor cell amplification. This amplification may explain the difference in the results obtained with this mycobacterium in various clinical immunotherapy trials in which it was applied differently. It is proposed to combine these mono- or pauc-functional adjuvants in order to try to obtain all of the beneficial effects of BCG without the amplification of suppressor cells.