The HPV-16 life cycle is strictly linked to the differentiation stage of the infected cell, and late viral mRNAs are expressed only in differentiated cells (14,37,43). Since all known HPV-16 promoters are located in one end of the viral genome, late gene expression is strongly influenced by posttranscriptional gene regulation (62). Viral RNA elements and cellular RNA binding factors are therefore important players in HPV-16 gene regulation (39, 45). The activities of these RNA elements and factors are responsible for the inhibition of HPV-16 late gene expression in proliferating cells and contribute to the induction of late gene expression in differentiating cells (45). RNA elements in the late untranslated region (UTR) of HPV-1 (50, 54, 56), HPV-16 (28), and HPV-31 (12) have been shown to inhibit late gene expression. These elements appear to be conserved among HPVs (60) and were originally identified in bovine papillomavirus (17). Late UTRs from different HPVs interact with the elav-like HuR protein (3, 48). Other RNA elements that suppress HPV-16 late gene expression have been identified previously. For example, the L1 and L2 coding regions contain inhibitory RNA elements that prevent the expression of L1 and L2 from subgenomic expression plasmids (10,11,42,49,53). The RNA elements in the L1 coding region were later shown to coincide with splicing silencers that suppress SA5639 (61), the only 3Ј splice site used exclusively by late mRNAs, whereas the RNA elements in the L2 coding region are active primarily as positive regulators of the early poly(A) signal pAE (41), thereby indirectly inhibiting late gene expression (41). Polyadenylation elements in L2 were originally identified in HPV-31 (55). In addition, splicing inhibitory sequences surround SD3632, the only 5Ј splice site used exclusively by late mRNAs, and therefore suppress late gene expression in proliferating cells (44). Finally, a major splicing enhancer downstream of the early 3Ј splice site SA3358 directs splicing to the early region of the genome and promotes polyadenylation at pAE (44), indirectly inhibiting late gene expression (44). Together, cellular factors interacting with these elements regulate HPV-16 gene expression. While we have previously identified hnRNP A1 as a cellular factor that binds to the splicing silencers in the HPV-16 L1 coding region and inhibits late mRNA splicing (61), most of the transacting factors regulating HPV-16 late gene expression remain to be identified. We have therefore initiated a screen for cellular factors that can induce HPV-16 late gene expression in proliferating cells. MATERIALS AND METHODSPlasmid constructions. pBEL, pBELM, pBSplice, and pBSpliceM have been described previously (pBSplice was referred to as pC16L1L2splice) (61). pT1, pT2, pT3, pT4, pT9, pT10, pT1OPSA, and pOPSDM have also been described previously (44), as have pBearly and pBELMDPU (59) and p1-22 M (58). pC16L1 and pC16L1M have been described previously as pC16L1MUT123 (11).

The role of CD28-mediated costimulation in secondary CD8+ T-cell responses remains controversial. Here, we have used two tools -blocking mouse anti-mouse CD28-specific antibodies and inducible CD28-deleting mice -to obtain definitive answers in mice infected with ovalbumin-secreting Listeria monocytogenes. We report that both blockade and global deletion of CD28 reveal its requirement for full clonal expansion and effector functions such as degranulation and IFN-γ production during the secondary immune response. In contrast, cell-intrinsic deletion of CD28 in transferred TCR-transgenic CD8 + T cells before primary infection leads to impaired clonal expansion but an increase in cells able to express effector functions in both primary and secondary responses. We suggest that the proliferation-impaired CD8 + T cells respond to CD28-dependent help from their environment by enhanced functional differentiation. Finally, we report that cell-intrinsic deletion of CD28 after the peak of the primary response does not affect the establishment, maintenance, or recall of long-term memory. Thus, if given sufficient time, the progeny of primed CD8 + T cells adapt to the absence of this costimulator. Keywords: CD28 costimulation CD8+ T cells CD8 + effector cell CD8 + T-cell memory Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionT-cell activation is controlled by the requirement for costimulation by the cell-surface receptor CD28 and its ligands CD80 and CD86, which are upregulated on professional APC as a result of innate pathogen recognition. Although some early studies showed that some primary T-cell responses can proceed in the absence of CD28Correspondence: Dr. Thomas Hünig e-mail: huenig@vim.uni-wuerzburg.de [1,2], it is now generally accepted that abolition of costimulation leads to limited immune responses, unless very strong or longlasting TCR stimulation is provided [3][4][5]. For memory responses, however, the importance of CD28-mediated costimulation is still controversial because the outcome of published studies varied with the methodology that was applied [6,7]. Early in vitro studies describe a CD28-independent expansion of CD8 + memory cells [5,8] which provide a functional immune system with regard to CD4 + T-cell help or regulatory T-cell functions. Alternatively, secondary responses of wild-type memory cells were studied after adoptive transfer into CD80 and CD86 knock-out mice, [9][10][11][12]. Deletion of CD80/86, however, does not only prevent ligation of CD28 but also of CTLA-4 (CD152), the inhibitory molecule on T cells that is upregulated during an active immune response to curtail the CD8 + T-cell response not only in a cell-intrinsic but also in a cell-extrinsic fashion, i.e. via its function as a suppressor molecule on regulatory T cells. This problem extends to the recently described interaction of another immune regulator, PD-L1, with these shared ligands (reviewed in [13]). Consequently, deleting the ligands of CD28 has a...

The costimulatory receptor CD28 and IL-4Rα-containing cytokine receptors play key roles in controlling the size and quality of pathogen-specific immune responses. Thus, CD28-mediated costimulation is needed for effective primary T-cell expansion and for the generation and activation of regulatory T-cells (Treg cells), which protect from immunopathology. Similarly, IL-4Rα signals are required for alternative activation of macrophages, which counteract inflammation by type 1 responses. Furthermore, immune modulation by CD28 and IL-4 is interconnected through the promotion of IL-4 producing T-helper 2 cells by CD28 signals. Using conditionally IL-4Rα and CD28 deleting mice, as well as monoclonal antibodies, which block or stimulate CD28, or mAb that deplete Treg cells, we have studied the roles of CD28 and IL-4Rα in experimental mouse models of virus (influenza), intracellular bacteria (L. monocytogenes, M. tuberculosis), and parasite infections (T. congolense, L. major). We observed that in some, but not all settings, Treg cells and type 2 immune deviation, including activation of alternative macrophages can be manipulated to protect the host either from infection or from immunopathology with an overall beneficial outcome. Furthermore, we provide direct evidence that secondary CD8 T-cell responses to i.c. bacteria are dependent on CD28-mediated costimulation.

Zusammenfassung Hintergrund Amblyopie ist der häufigste Grund einer Sehminderung im Kindesalter. Wichtige Risikofaktoren für eine Amblyopie (ARF) sind Refraktionsfehler. Ziel dieser Studie war zu untersuchen, wie viele Kinder mit auffälligem Screening eine Untersuchung beim Augenarzt erhielten und wie verlässlich der Plusoptix Autorefractor A09 (POA09) in der Ermittlung refraktiver ARF ist. Methodik Wir führten eine prospektive, einarmige und nicht verblindete Studie von 2/2012 bis 9/2015 durch. Es wurden Kinder im Alter von 6 Monaten bis 12 Jahren in Kindergärten und Schulen auf refraktive ARF gescreent. Folgende Refraktionswerte galten im Screening und bei der Messung des Refraktionsfehlers in Zykloplegie als auffällig: Hyperopie ≥ 3,5 Dioptrien (D), Myopie ≥ 3,0 D, Anisometropie ≥ 1,5 D und Astigmatismus ≥ 1,5 D (90° oder 180° ± 10°) oder ≥ 1,0 D (≥ 10° Abweichung von 90° oder 180°). Im Screening auffälligen Kindern wurde eine Vorstellung beim Augenarzt empfohlen, und deren Sorgeberechtigte wurden hinsichtlich der Ergebnisse des Augenarztbesuches nachbefragt. Kinder mit auffälligem Screening sowie eine Referenzgruppe aus Kindern mit unauffälligem Screening erhielten eine vollständige orthoptische Untersuchung und eine Messung des Refraktionsfehlers in Zykloplegie. Anhand der Kinder mit auffälligem Screening wurde der Anteil der richtig erkannten refraktiven ARF untersucht. Anhand der Referenzgruppe wurden der Anteil der richtig ausgeschlossenen refraktiven ARF und die falsch negative Rate berechnet. Ergebnisse Es wurden 3170 Kinder gescreent. Bei 715 Kindern (22,3 %) war das Screening auffällig, von diesen lag bei 460 (64,3 %) Antwort auf die Nachbefragung vor, und bei 132 lagen vollständige Angaben zu in Zykloplegie gemessenen Refraktionsfehlern vor. Häufigste Auffälligkeiten im Screening waren Astigmatismus (90,9 %) und Anisometropie (11,4 %). Nach Messung des Refraktionsfehlers in Zykloplegie waren Astigmatismus (56,8 %) und Hyperopie (18,9 %) am häufigsten. Der Anteil im Screening richtig erkannter refraktiver ARF war für Astigmatismus (60 %) und Anisometropie (53,3 %) am höchsten, für Hyperopie (33,3 %) und Myopie (25 %) geringer. Schlussfolgerung Refraktive ARF konnten im Screening mit dem POA09 nur eingeschränkt erkannt werden, was die Wichtigkeit eines systematischen Amblyopiescreenings unterstreicht. Denkbar wäre u. a. ein Screening in Zykloplegie, wozu weitere Studien erforderlich sind.