NKG2D receptor signaling shapes T cell thymic education.

The role of natural killer group 2D (NKG2D) in peripheral T cells as a costimulatory receptor is well established. However, its contribution to T cell thymic education and functional imprint is unknown. Here, we report significant changes in development, receptor signaling, transcriptional program, and function in T cells from mice lacking NKG2D signaling. In C57BL/6 (B6) and OT-I mice, we found that NKG2D deficiency results in Vß chain usage changes and stagnation of the double-positive stage in thymic T cell development. We found that the expression of CD5 and CD45 in thymocytes from NKG2D deficient mice were reduced, indicating a direct influence of NKG2D on the strength of T cell receptor (TCR) signaling during the developmental stage of T cells. Depicting the functional consequences of NKG2D, peripheral OT-I NKG2D-deficient cells were unresponsive to ovalbumin peptide stimulation. Paradoxically, while αCD3/CD28 agonist antibodies led to phenotypic T cell activation, their ability to produce cytokines remained severely compromised. We found that OT-I NKG2D-deficient cells activate STAT5 in response to interleukin-15 but were unable to phosphorylate ERK or S6 upon TCR engagement, underpinning a defect in TCR signaling. Finally, we showed that NKG2D is expressed in mouse and human thymic T cells at the double-negative stage, suggesting an evolutionarily conserved function during T cell development. The data presented in this study indicate that NKG2D impacts thymic T cell development at a fundamental level by reducing the TCR threshold and affecting the functional imprint of the thymic progeny. In summary, understanding the impact of NKG2D on thymic T cell development and TCR signaling contributes to our knowledge of immune system regulation, immune dysregulation, and the design of immunotherapies.

Targeting intracellular oncoproteins with dimeric IgA promotes expulsion from the cytoplasm and immune-mediated control of epithelial cancers.

Dimeric IgA (dIgA) can move through cells via the IgA/IgM polymeric immunoglobulin receptor (PIGR), which is expressed mainly on mucosal epithelia. Here, we studied the ability of dIgA to target commonly mutated cytoplasmic oncodrivers. Mutation-specific dIgA, but not IgG, neutralized KRASG12D within ovarian carcinoma cells and expelled this oncodriver from tumor cells. dIgA binding changed endosomal trafficking of KRASG12D from accumulation in recycling endosomes to aggregation in the early/late endosomes through which dIgA transcytoses. dIgA targeting of KRASG12D abrogated tumor cell proliferation in cell culture assays. In vivo, KRASG12D-specific dIgA1 limited the growth of KRASG12D-mutated ovarian and lung carcinomas in a manner dependent on CD8+ T cells. dIgA specific for IDH1R132H reduced colon cancer growth, demonstrating effective targeting of a cytoplasmic oncodriver not associated with surface receptors. dIgA targeting of KRASG12D restricted tumor growth more effectively than small-molecule KRASG12D inhibitors, supporting the potential of this approach for the treatment of human cancers.

Sézary syndrome originates from heavily mutated hematopoietic progenitors.

The pathogenesis of cutaneous T-cell lymphoma (CTCL) remains unclear. Using single-cell RNA or T-cell receptor (TCR) sequencing of 32 619 CD3+CD4+ and CD26+/CD7+ and 29 932 CD3+CD4+ and CD26-/CD7- lymphocytes from the peripheral blood of 7 patients with CTCL, coupled to single-cell ATAC-sequencing of 26,411 CD3+CD4+ and CD26+/CD7+ and 33 841 CD3+CD4+ and CD26-/CD7- lymphocytes, we show that tumor cells in Sézary syndrome and mycosis fungoides (MF) exhibit different phenotypes and trajectories of differentiation. When compared to MF, Sézary cells exhibit narrower repertoires of TCRs and exhibit clonal enrichment. Surprisingly, we identified ≥200 mutations in hematopoietic stem cells from multiple patients with Sézary syndrome. Mutations in key oncogenes were also present in peripheral Sézary cells, which also showed the hallmarks of recent thymic egression. Together our data suggest that CTCL arises from mutated lymphocyte progenitors that acquire TCRs in the thymus, which complete their malignant transformation in the periphery.

Actionable spontaneous antibody responses antagonize malignant progression in ovarian carcinoma.

OBJECTIVE: To demonstrate that shared antibody responses in endometriosis and endometriosis-associated ovarian cancer spontaneously antagonize malignant progression and can be leveraged to develop future immunotherapies. METHODS: B cells from cyopreserved clear cell ovarian carcinoma (CCC, n = 2), endometrioid ovarian carcinoma (EC, n = 2), and endometriomas (n = 2) were isolated, activated, and EBV-immortalized. Antibodies were purified from B cell supernatants and used for screening arrays containing most of the human proteome. Targets were prioritized based on accessibility (transmembrane or secreted proteins), expression in endometriosis and cancer, and concurrent IgA and IgG responses. We focused on antibodies targeting tumor-promoting syndecan binding protein (SDCBP) to demonstrate anti-tumor activity. Immunoblots and qPCR were performed to assess SDCBP expression in ovarian cancer and endometriosis cell lines and tumor samples. Recombinant IgG4 was generated using the variable heavy and light chains of dominant B cell receptors (BCRs) reacting against the extracellular domain of SDCBP, and used in in vivo studies in human CCC- and high-grade serous ovarian carcinoma (HGSOC)-bearing immunodeficient mice. RESULTS: Nine accessible proteins detected by both IgA and IgG were identified in all samples - including SDCBP, which is expressed in ovarian carcinomas of multiple histologies. Administration of α-SDCBP IgG4 in OVCAR3 (HGSOC), TOV21G and RMG-I (CCC) tumor-bearing mice significantly decreased tumor volume compared to control irrelevant IgG4. CONCLUSIONS: Spontaneous antibody responses exert suboptimal but measurable immune pressure against malignant progression in ovarian carcinomas. Using tumor-derived antibodies for developing novel immunotherapeutics warrants further investigation.

Anti-4-1BB immunotherapy enhances systemic immune effects of radiotherapy to induce B and T cell-dependent anti-tumor immune activation and improve tumor control at unirradiated sites.

Radiation therapy (RT) can prime and boost systemic anti-tumor effects via STING activation, resulting in enhanced tumor antigen presentation and antigen recognition by T cells. It is increasingly recognized that optimal anti-tumor immune responses benefit from coordinated cellular (T cell) and humoral (B cell) responses. However, the nature and functional relevance of the RT-induced immune response are controversial, beyond STING signaling, and agonistic interventions are lacking. Here, we show that B and CD4+ T cell accumulation at tumor beds in response to RT precedes the arrival of CD8+ T cells, and both cell types are absolutely required for abrogated tumor growth in non-irradiated tumors. Further, RT induces increased expression of 4-1BB (CD137) in both T and B cells; both in preclinical models and in a cohort of patients with small cell lung cancer treated with thoracic RT. Accordingly, the combination of RT and anti-41BB therapy leads to increased immune cell infiltration in the tumor microenvironment and significant abscopal effects. Thus, 4-1BB therapy enhances radiation-induced tumor-specific immune responses via coordinated B and T cell responses, thereby preventing malignant progression at unirradiated tumor sites. These findings provide a rationale for combining RT and 4-1bb therapy in future clinical trials.

Memory CD4 T cell-derived IL-2 synergizes with viral infection to exacerbate lung inflammation.

Defining the most penetrating correlates of protective memory T cells is key for designing improved vaccines and T cell therapies. Here, we evaluate how interleukin (IL-2) production by memory CD4 T cells, a widely held indicator of their protective potential, impacts immune responses against murine influenza A virus (IAV). Unexpectedly, we show that IL-2-deficient memory CD4 T cells are more effective on a per cell basis at combating IAV than wild-type memory cells that produce IL-2. Improved outcomes orchestrated by IL-2-deficient cells include reduced weight loss and improved respiratory function that correlate with reduced levels of a broad array of inflammatory factors in the infected lung. Blocking CD70-CD27 signals to reduce CD4 T cell IL-2 production tempers the inflammation induced by wild-type memory CD4 T cells and improves the outcome of IAV infection in vaccinated mice. Finally, we show that IL-2 administration drives rapid and extremely potent lung inflammation involving NK cells, which can synergize with sublethal IAV infection to promote acute death. These results suggest that IL-2 production is not necessarily an indicator of protective CD4 T cells, and that the lung environment is particularly sensitive to IL-2-induced inflammation during viral infection.

Heat capacities of dipolar fluids: ferromagnetic colloids.

The heat capacities of ferrofluids are investigated using a thermodynamic perturbation theory approach and the NVT and NpT Monte Carlo simulation methods. The systems studied are considered as one-, three-, and five-component dipolar mixtures modeled by the Stockmayer interaction potential. The isochoric and isobaric heat capacities are calculated and compared with the data determined for a monodisperse equivalent of the system.

UV-B radiation induced exchange of the D1 reaction centre subunits produced from the psbA2 and psbA3 genes in the Cyanobacterium synechocystis sp. PCC 6803.

UV-B irradiation of Synechocystis 6803 cells inhibits photosystem II activity, which can be restored via de novo synthesis of the D1 (and D2) reaction center subunits. Recently we have shown that of the two psbA genes that encode identical D1 proteins in Synechocystis 6803, UV-B preferentially enhances the transcription of psbA3 compared to that of psbA2 [Máté, Z., Sass, L., Szekeres, M., Vass, I. and Nagy, F. (1998) J. Biol. Chem. 273, 17439-17444]. Here we studied the effect of UV-B on the synthesis of the D1 protein from the psbA2 and psbA3 genes in the P7 mutant of Synechocystis 6803. In this mutant, psbA2 carries the Ala251-->Val point mutation, which confers resistance to the photosystem II electron transport inhibitor metribuzin, but psbA3 is the same as in the wild-type. By applying variable chlorophyll fluorescence measurements to distinguish between metribuzin-sensitive and metribuzin-resistant photosystem II centers we quantified the amount of the D1 protein produced from each of the psbA3 and psbA2 genes. When the cells were exposed to UV-B light, the fraction of D1 protein produced from the psbA3 gene was increased from 15-20 to 32-40% of the total D1. This effect was reversible by transferring the cells to visible light. The rate of D1 production from psbA3 increased with increasing UV-B intensities, and was a transient phenomenon at low UV-B levels (0.1 microE x m-2 x s-1). It is concluded that the enhancement of psbA3 gene transcription by UV-B light leads to enhanced D1 protein synthesis from this gene. Our findings demonstrate that the main role of psbA3 transcription activated by UV-B is to increase the size of the psbA mRNA pool available for translation when a rapid repair of the D1 protein is needed under UV-B stress.

UV-B induced differential transcription of psbD genes encoding the D2 protein of Photosystem II in the cyanobacterium Synechocystis 6803.

UV-B irradiation of intact Synechocystis sp. PCC 6803 cells results in the loss of Photosystem II activity, which can be repaired via de novo synthesis of the D1 and D2 reaction center subunits. A key step in the repair process is the differential transcription of the psbA2 and psbA3 genes, coding for identical D1 polypeptides [Máté et al. (1998) J Biol Chem 273: 17439-17444]. In the present work, we investigated for the first time the effect of UV-B irradiation on the transcription of the psbD1 and psbD2 genes encoding identical D2 polypeptides. By using gene-specific S1 nuclease protection assay we showed differential UV-B induced transcription of the two psbD genes: the level of psbD1 mRNA was increased 1.5-2 fold, whereas the accumulation of psbD2 mRNA was 5-7 fold. The induction of psbD2 transcript accumulation by low intensity light was specific for the UV-B range. UV-A emission from the applied UV source, as well as 100 muE m(-2) s(-1) white light had negligible effect. Increase in the psbD2 mRNA level was observed at very low UV-B intensities, which did not cause damage to the function and protein structure of PS II. Expression patterns of chimeric genes containing the promoter regions of the psbD1, psbD2 genes fused to the firefly luciferase (luc) reporter gene showed similar induction as observed for the endogenous psbD genes. Our findings demonstrate that UV-B radiation induces differential expression of the of the psbD1 and psbD2 genes. We propose that the primarily expressed psbD2 serves as a UV stress gene and participates in a rapid defense response against UV-B stress. This effect is regulated, at least partially, at the level of transcription.

Prevalence of bovine herpesvirus 1 (BHV-1) infection in Hungarian cattle herds.

Hungarian cattle herds were surveyed for bovine herpesvirus 1 (BHV-1) infection by ELISA of milk and serum samples. In 1993, 75% of the large cattle herds (consisting of more than 50 cattle) and all small herds (small-scale producers' stocks), while in 1997 90% of the small herds were included in the survey. In the case of large herds, 79.3% of the herds and 64.1% of the samples tested were found to be positive. Of the small herds, 13.5% and 15.7% tested positive in 1993 and 1997, respectively. The majority of large herds were Holstein-Friesian dairy stocks. Small herds with an infection rate markedly exceeding the average were found in those counties where the small herds had been in close contact with the large-scale farms, or where new herds were established by using animals of uncontrolled infectious bovine rhinotracheitis (IBR) status originating from large farms. Attention is called to the importance of maintaining the IBR-free status of small herds that constitute one-third of the Hungarian cattle population.

UV-B-induced differential transcription of psbA genes encoding the D1 protein of photosystem II in the Cyanobacterium synechocystis 6803.

UV-B irradiation of intact Synechocystis sp. PCC 6803 cells results in the loss of photosystem II activity, which can be repaired via de novo synthesis of the D1 (and D2) reaction center subunits. In this study, we investigated the effect of UV-B irradiation on the transcription of the psbA2 and psbA3 genes encoding identical D1 proteins. We show that UV-B irradiation increases the level of psbA2 mRNA 2-3-fold and, more dramatically, it induces a 20-30-fold increase in the accumulation of the psbA3 mRNA even at levels of irradiation too low to produce losses of either photosystem II activity or D1 protein. The induction of psbA3 transcript accumulation is specific for UV-B light (290-330 nm). Low intensity UV-A emission (330-390 nm) and white light induce only a small, at most, 2-3-fold enhancement, whereas no effect of blue light was observed. Expression patterns of chimeric genes containing the promoter regions of the psbA2, psbA3 genes fused to the firefly luciferase (luc) reporter gene indicate that (i) transcription of psbA2/luc and psbA3/luc transgenes was elevated, similarly to that of the endogenous psbA genes, by UV-B irradiation, and that (ii) a short, 80-base pair psbA3 promoter fragment is sufficient to maintain UV-B-induced transcription of the luc reporter gene. Furthermore, our findings indicate that UV-B-induced expression of the psbA2 and psbA3 genes is a defense response against UV-B stress, which is regulated, at least, partially at the level of transcription and does not require active electron transport.

Resistance of reaction centers from Rhodobacter sphaeroides against UV-B radiation. Effects on protein structure and electron transport.

Inhibition of electron transport and damage to the protein subunits by ultraviolet-B (UV-B, 280-320 nm) radiation have been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides R26. UV-B irradiation results in the inhibition of charge separation as detected by the loss of the initial amplitude of absorbance change at 430 nm reflecting the formation of the P(+)(QAQB)(-) state. In addition to this effect, the charge recombination accelerates and the damping of the semiquinone oscillation increases in the UV-B irradiated reaction centers. A further effect of UV-B is a 2 fold increase in the half- inhibitory concentration of o-phenanthroline. Some damage to the protein subunits of the RC is also observed as a consequence of UV-B irradiation. This effect is manifested as loss of the L, M and H subunits on Coomassie stained gels, but not accompanied with specific degradation products. The damaging effects of UV-B radiation enhanced in reaction centers where the quinone was semireduced (QB (-)) during UV-B irradiation, but decreased in reaction centers which lacked quinone at the QB binding site. In comparison with Photosystem II of green plant photosynthesis, the bacterial reaction center shows about 40 times lower sensitivity to UV-B radiation concerning the activity loss and 10 times lower sensitivity concerning the extent of reaction center protein damage. It is concluded that the main effect of UV-B radiation in the purple bacterial reaction center occurs at the QAQB quinone acceptor complex by decreasing the binding affinity of QB and shifting the electron equilibration from QAQB (-) to QA (-)QB. The inhibitory effect is likely to be caused by modification of the protein environment around the QB binding pocket and mediated by the semiquinone form of QB. The UV-resistance of the bacterial reaction center compared to Photosystem II indicates that either the QAQB acceptor complex, which is present in both types of reaction centers with similar structure and function, is much less susceptible to UV damage in purple bacteria, or, more likely, that Photosystem II contains UV-B targets which are more sensitive than its quinone complex.

Combined therapy with isoprinosine and CO2 laser microsurgery for the treatment of laryngeal papillomatosis.

Recurrent laryngeal papillomas and their potential malignant degenerations in adult patients have been known clinically for about 100 years. An early effective treatment has great importance in preventing possible obstruction of the airway or malignant change. The use of endoscopic microsurgery with the CO2 laser has resulted in a significant improvement in the eradication of papillomatous nodules. The advantages of laser surgery are: better visualization of the larynx to allow more precise and deeper resection of papillomas, limited bleeding, and a reduced possibility of seeding uninvolved mucosa. Although laser surgery is the current method of choice for treating laryngeal papillomatosis, it has not solved completely the problems of recurrences. To make treatment more effective, we have developed a therapeutic regimen that combines laser microsurgery with the immunostimulant methisoprinol (Isoprinosine). Our observations in 18 patients have shown that this combined management was more successful than using a single modility with either the laser or Isoprinosine alone. The combined approach was also effective in recurrent cases, with the timing of the combination influencing the results of the treatment given.