TCR sequencing: applications in immuno-oncology research.

•T-cell receptor (TCR) interaction with major histocompatibility complex-antigen complexes leads to antitumour responses.•TCR sequencing analysis allows characterisation of T cells that recognise tumour neoantigens.•T-cell clonal revival and clonal replacement potentially underpin immunotherapy responses.

Conventional protein kinase C plays a critical role in negative regulation of CD98-induced homotypic aggregation.

CD98, a heterodimeric type II transmembrane protein, is involved in many different cellular events, ranging from amino acid transport to cell-cell adhesion. Little is known about the positive and negative signalling pathways involved in these responses. Therefore, we examined the role of conventional protein kinase C (PKC) isoforms during CD98-induced intracellular signalling and homotypic aggregation of U937 cells. The CD98-induced aggregation was enhanced by the general protein kinase inhibitors GF109203X and staurosporin, and by specific PKC-alpha/-beta peptide inhibitor 19-27, but inhibited by PKC activators such as phorbol 12-myristate 13-acetate (PMA). PMA-inhibition was reversed by PKC inhibitors recognising the ATP-binding site in PKC (e.g. staurosporin, GF109203X and Go6983). Inhibitors which bind to diacylglycerol (DAG) or Ca(2+)-binding sites of PKC (calphostin C and Go6967) had no effect. PMA-induced translocation of conventional PKC (cPKC) isozymes (alpha, beta and gamma), but decreased the expression of PKC-delta, which plays an important role in CD98-induced homotypic aggregation. PMA treatment also suppressed the surface level of CD98 but not CD29, CD18 and CD147, dose- and time-dependently. These data provide evidence that PMA-responsive cPKC isoforms (alpha, beta and gamma) play a key role in negative regulation of CD98 signalling and homotypic aggregation.

Isolation and characterization of a new murine MHC class II transcription mutant cell line.

We have isolated and characterized a new MHC class II transcription mutant cell line, called UV. This cell line was derived from the mouse B lymphoma A20 by UV light-induced mutagenesis and immunoselection for the loss of surface MHC class II molecules. It expresses only 5% of the level of MHC class II molecules on A20 and this is associated with a similar reduction of class II specific mRNA. This defect cannot be restored by the MHC class II transcription inducers, IL-4 and IFN gamma, confirming that the mutation acts at the transcription level. The mutation also affects MHC class I expression, but the transcription of class I molecules is not affected. In contrast, the expression of other markers, such as the invariant chain and the surface immunoglobulins G and M, is not modified. Such a variant should prove useful for the study of the transcription factors involved in the regulation of MHC class II expression.

Isolation and N-terminal sequence determination of a novel gamma/delta T cell surface antigen.

An antigen complex unique for porcine gamma/delta T cells has previously been identified using the monoclonal antibodies MAC319 and MAC320. Here we use digestion with the proteolytic enzyme bromelain to selectively release the MAC319 antigen as a soluble fragment, for further characterisation. A cytofluorometric inhibition assay was developed to follow the purification of this fragment, as the conformation sensitivity of the MAC319 epitope prevented the use of immunoblotting techniques. The antigen has been purified using a combination of anion-exchange and hydrophobic-interaction columns, followed by separation on a size-exclusion column. Fractions from the size-exclusion column containing the antigen consisted of one major band at Mr 95,000. This species was shown to be specifically absorbed onto MAC319-coupled Sepharose, thereby identifying the MAC319 antigen. N-terminal amino acid sequencing of this band has revealed a previously unidentified sequence. This fragment was also shown to be glycosylated, most likely with a single sugar moiety. Enzymatic removal of the sugars showed that they did not appear to be necessary for binding of the polypeptide to the MAC319 antibody.

The role of reactive oxygen species in triggering proliferation and IL-2 secretion in T cells.

This paper examines the hypothesis that reactive oxygen species (ROS) play an important role as second messengers in T cell activation. Activation of T cells with phorbol ester in combination with either calcium ionophore, or anti-CD3 antibody results in a large rapid flux of ROS activity. In contrast, co-stimulation with CD28 does not enhance ROS activity. The ROS signal was sensitive to ascorbic acid, desferrioxamine and dimethyl sulfoxide, suggesting that the major active species being generated was the hydroxyl radical, probably by iron-catalyzed decomposition of hydrogen peroxide. The generation of ROS in T cells was regulated by an accessory population within the peripheral blood. An anti-CD2 antibody induced a strong ROS flux, suggesting that the CD2/LFA-3 interaction may be important in this regulation. T cell activation was inhibited by the same panel of anti-oxidants as ROS generation, but much higher concentrations were required for inhibition of proliferation and IL-2 release than those required to block ROS generation. These data imply that ROS are not obligate second messengers for initiation of T cell activation. The results are compatible, however, with a role for activation-dependent T cell ROS generation in modulating the overall T cell response via autocrine and paracrine signalling pathways.

Reactive oxygen species activate human peripheral blood dendritic cells.

This study investigates the effects of hydrogen peroxide, a potent oxygen free radical donor, on the phenotype and function of dendritic cells differentiated from peripheral blood precursors. We report that hydrogen peroxide induces an up-regulation of several dendritic cell surface markers involved in interaction with T cells, including MHC Class II molecules (DQ and DR) and the co-stimulatory molecules CD40 and CD86. Moreover we have observed that H2O2-treated dendritic cells are more efficient in promoting T cell proliferation than normal dendritic cells and that this enhancement can be blocked using the free radical scavenger agent N-acetylcysteine. Oxygen free radicals are a common by-product of inflammation, and our results suggest they may play an important role in activation of sentinel dendritic cells, linking tissue damage to the initiation of an adaptive immune response.

Improvement of the in vitro T cell proliferation assay by a modified method that separates the antigen recognition and IL-2-dependent steps.

T cell activation is commonly assayed in vitro by measuring the proliferative response of primed cells to an antigenic stimulus. We have modified the conventional form of this assay by dividing up the response into two stages. During the first stage, antigen drives the specific expression of IL-2 receptor expression. This phase is carried out in the presence of homologous mouse serum, in order to reduce non-specific responses to a minimum. During the second phase, proliferation of these activated T cells is driven by the addition of excess exogenous IL-2. This modified form of proliferation assay significantly increased the signal to noise ratio which can be attained, and is of particular value when looking at the T cell response to weak (e.g., cross-reactive) antigens, or low concentrations of antigen.

Observations on the cytochemistry of the hemocytes of an insect, Galleria mellonella.

A number of cytochemical parameters of the hemocytes of larval Galleria mellonella, an insect frequently used as a model by comparative cellular immunologists, are described. Cytochemical methods were used to quantify hemocyte granule-associated components, the results are compared to those obtained for leukocytes from higher animals. Granulocytes contained a population of nonlysosomal granules rich in mucopolysaccharide not seen in plasmatocytes. The numbers and dimensions of these granules showed a positive correlation to cell size, probably reflecting a developmental sequence in granulocyte maturation. Both granulocytes and plasmatocytes had other granules containing the typical lysosomal enzymes, acid phosphatase, beta-glucuronidase, esterase, and lysozyme. The nonlysosomal enzyme alkaline phosphatase was not found in Galleria hemocytes; it is also absent from vertebrate monocytes, macrophages, and immature polymorphonuclear leukocytes. Insect hemocytes appear to lack certain components of antibacterial systems typical of mammalian blood cells, such as H2O2-generating systems, cationic proteins, and myeloperoxidase. The bactericidal mechanisms of hemocytes probably involve lysozyme, as well as other biologically active cellular and humoral factors unique to insects.

Sensitization of allo-specific T lymphocytes in vivo: role of antigen-presenting cells.

The migratory behavior of antigen-presenting cells was investigated in vivo. Purified murine splenic dendritic cells and splenic and peritoneal macrophages were labelled and injected subcutaneously in the hind foot-pads of mice and monitored for seven days. In the first 24 h, a small quantity of label was recovered from popliteal but not inguinal lymph nodes with radioactive (111In-oxine and 3H-uridine) but not fluorescent (1,1'-dioctadecyl 3,3,3'3'-tetramethylindocarbocyanine perchlorate and fluorescein isothiocyanate) labelling of the antigen-presenting cells. Chemical fixation of the injected antigen-presenting cells had no effect on the detection of label in the popliteal lymph nodes, suggesting that it was unlikely to be due to active cellular migration. Label recovery from hind feet declined with time over the seven day period and was independent of the label type. Essentially the same observations were made whether the antigen-presenting cells were syngeneic or allogeneic to the injected mice and irrespective of the type of antigen-presenting cell used. However, allogeneic antigen-presenting cells, which did not migrate to the draining lymph nodes, successfully primed T lymphocytes in these lymph nodes as shown by a secondary in vitro mixed leukocyte reaction. Again, chemical fixation of the injected antigen-presenting cells had no effect on their ability to prime allogeneic T lymphocytes in the draining lymph nodes. These experiments suggest that, during experimental allo-sensitization via the subcutaneous route, indirect priming of allogeneic T lymphocytes may be a dominant pathway.

Prostaglandin E2 (PGE2) differentially regulates the production of IL-2 and IL-3 by murine immune T-cells.

Eicosanoids are important mediators of inflammation, and have been shown to have potent, and usually suppressive immunoregulatory activities. In the paper, we have examined the role of prostaglandin (PGE2) production in the regulation of two cytokines, IL-2 and IL-3, which both play a key role in contact sensitivity and delayed type hypersensitivity reactions. In agreement with previous studies, we demonstrate that prostaglandins down-regulate IL-2 production in the system. Unexpectedly, however, IL-3 levels are enhanced in the presence of the prostaglandin PGE2 and conversely, are inhibited by treatment with aspirin, a potent inhibitor of prostaglandin metabolism. The implications of this result in terms of the immunoregulatory role of PGs will be discussed.

Blood cells contribute to glial repair in an insect.

Using antibodies specific for haemocytes, we have shown that these blood cells penetrate the abdominal nervous connectives of the cockroach following selective disruption of the glia using the DNA-intercalating drug, ethidium bromide, as a glial toxin. Within 4 days post-lesion, the labelled cells formed a mosaic beneath the neural lamella and penetrated deeply among the disrupted subperineurial glia. These observations confirm that exogenous cells are involved in glial repair and support a previous hypothesis that they play critical roles in both structural repair and the recruitment of endogenous reactive cells.

Characterization of soluble factors from cultures of premalignant cervical epithelium.

PURPOSE: Immune responses within the cervical microenvironment are likely to play an important role in the natural history of premalignant lesions but the pattern of this response and how it is regulated has not been documented in detail. METHODS: Explants of premalignant cervical epithelium were cultured in vitro for 24 hours. The culture supernatants were assayed for the presence of IL-1alpha, IL-10, IL-12 and TNF-alpha by ELISA. Aliquots of each supernatant were also added to a CD3-dependent T cell proliferation assay. RESULTS: The pattern of cytokines found in different samples was heterogeneous and no significant correlation was observed between the various cytokines examined. The functional effects observed were also diverse, with some supernatants showing strong inhibitory T cell activity, while others were stimulatory. CONCLUSION: Our results document the heterogeneity of the local cytokine microenvironment of premalignant cervical lesions, which may play a role in regulating the immune response associated with such lesions and hence influence clinical outcome.

Autocrine type I interferon amplifies dendritic cell responses to lipopolysaccharide via the nuclear factor-kappaB/p38 pathways.

The central role of dendritic cells (DC) in the initiation of immune responses requires these cells to be able to determine the degree of danger in their microenvironment. Abrogating the activity of type I interferon (IFN) secreted after lipopolysaccharide (LPS) stimulation of DC inhibits CD86 and human leucocyte antigen-DR (HLA-DR) upregulation at a low LPS concentration. At a higher concentration of LPS, while changes in surface phenotype are not dependent on type I IFN, this cytokine is required for maximal secretion of interleukin-12 (IL-12) and tumour necrosis factor-alpha (TNFalpha) by DC. Thus, the secretion and autocrine activity of type I IFN after Toll-like receptor stimulation enables DC to orchestrate a hierarchical maturation response with regard to changes in surface phenotype and secretion of cytokines. In addition, the activation of nuclear factor-kappaB and p38 pathways in DC can occur either in an additive fashion when DC are exposed to dual stimulation or can be activated in discrete phases over time when DC are exposed to LPS alone. The differential activation of these pathways provides a mechanism for DC to integrate the activation by multiple stimuli and thus amplify responses to pathogen infection.

The interaction between staphylococcal superantigen-like proteins and human dendritic cells.

Staphylococcus aureus produce a family of exotoxins (staphylococcal superantigen like proteins, SSLs) with structural, but not functional, homology to superantigens. These proteins have previously been shown to interact selectively with antigen presenting cells, including dendritic cells. The functional consequences of this interaction are now explored. SSL7 and 9 had no effect on viability or morphology of dendritic cells. The proteins did not induce dendritic cell maturation, as measured by cell surface phenotype. Exposure to SSL did not alter the ability of dendritic cells to take up FITC-dextran. Finally, exposure to SSLs did not impair the ability of the dendritic cells to stimulate allogeneic or antigen specific T cell responses. However, dendritic cells loaded with SSL7 or 9 were able to stimulate a T cell proliferative response in 3/8 healthy individuals tested. Sera from nine out of 10 individuals tested contained antibodies against both SSL7 and SSL9, and the response to each SSL was specific and not cross-reactive. The results demonstrate that SSLs are immunogenic in humans at both the B and T cell level, but it remains unclear whether this response is to the benefit of the bacterium or the host.

Penicillin and beyond.

The discovery of penicillin remains one of the greatest advances in medical science. From the success of the discovery the biotechnology industry became established.

Analysis of the cellular requirements for the binding of exogenous peptides to MHC class II molecules.

The cellular mechanism regulating the binding of exogenous peptides to MHC class II molecule is still an object of controversy. In order to study the cellular requirements of peptide binding we have set up an indirect fluorescence assay that enables us to detect quantitatively peptide/MHC class II complexes on the cell surface of the mouse B lymphoma A20. The absence of binding on several MHC class II-negative cell lines and the inhibition of binding in the presence of competitor peptides or in the presence of a polyclonal serum against MHC class II molecules confirmed the specificity of the assay. A panel of pharmacological and physical agents was then used to determine the mechanism of this regulation. Binding was not significantly affected by vinblastine or cycloheximide and was affected only to a small extent by chloroquine or azide. In contrast to the long half-life previously reported for soluble complexes, we found that the half-life of a peptide/MHC class II complex expressed on A20 was shorter than 3 hr, suggesting that peptide binding might be regulated at the cellular level. The energy of activation of peptide binding, estimated from the temperature dependence of the rate of peptide binding, was decreased above 27 degrees C, suggesting that enhanced peptide binding to MHC class II molecules might depend on the fluidity of the cell membrane lipids.

Antigen-specific inhibition of IL-2 and IL-3 production in contact sensitivity to TNP.

The production of IL-2 and IL-3 by T cells from mice which had been contact sensitized to TNP and/or tolerized by intravenous injections of TNBS was assayed. Contact sensitization rapidly primes T cells, so that they respond to in vitro restimulation with haptenated syngeneic cells by producing IL-2 and IL-3. This production is strongly inhibited, in an antigen-specific manner, in tolerized mice. At least part of this inhibition can be attributed to the action of suppressor T cells that act by preventing the activation of lymphokine production in vitro. Lymphokine production thus closely parallels the in vivo delayed-type hypersensitivity (DTH) reaction in this system.