Cooperation between CD4 and CD8 T cells for anti-tumor activity is enhanced by OX40 signals

The relative contribution of OX40 (CD134) to priming of CD8 T cells in complex systems where CD4 and CD8 cells respond and cooperate together is not clear. We previously found that OX40 expressed on tumor-reactive CD8 T cells controls their initial persistence when adoptively transferred in vivo and is required for delayed tumor growth. We now show that exogenous stimulation of OX40 with agonist antibody augments its ability to suppress the growth of new as well as established tumors, correlating with marked expansion of adoptively transferred CD8 T cells. Concomitantly, anti-OX40 strongly enhanced the number of tumor antigen-reactive CD4 T cells. Moreover, the augmented accumulation of CD8 T cells was prevented in animals lacking MHC class II or depleted of CD4 cells and did not occur in OX40-deficient animals receiving wild-type CD8 cells, demonstrating that non-CD8 cells are the major target of OX40 signals. These results suggest that while OX40 signaling to a CD8 T cell can control its expansion, OX40 expressed on non-CD8 cells strongly influences CD8 priming and in vivo activity. OX40 therefore represents an important signal for allowing effective cooperation between CD4 and CD8 cells and for promoting cell interplay and tumor rejection where CD8 activity is limiting.     

Interactions of antagonists with subtypes of inositol 1,4,5-trisphosphate (IP3) receptor

BACKGROUND AND PURPOSE

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are intracellular Ca²⁺ channels. Interactions of the commonly used antagonists of IP₃Rs with IP₃R subtypes are poorly understood.

EXPERIMENTAL APPROACH

IP₃-evoked Ca²⁺ release from permeabilized DT40 cells stably expressing single subtypes of mammalian IP₃R was measured using a luminal Ca²⁺ indicator. The effects of commonly used antagonists on IP₃-evoked Ca²⁺ release and ³H-IP₃ binding were characterized.

KEY RESULTS

Functional analyses showed that heparin was a competitive antagonist of all IP₃R subtypes with different affinities for each (IP₃R3 > IP₃R1 ≥ IP₃R2). This sequence did not match the affinities for heparin binding to the isolated N-terminal from each IP₃R subtype. 2-aminoethoxydiphenyl borate (2-APB) and high concentrations of caffeine selectively inhibited IP₃R1 without affecting IP₃ binding. Neither Xestospongin C nor Xestospongin D effectively inhibited IP₃-evoked Ca²⁺ release via any IP₃R subtype.

CONCLUSIONS AND IMPLICATIONS

Heparin competes with IP₃, but its access to the IP₃-binding core is substantially hindered by additional IP₃R residues. These interactions may contribute to its modest selectivity for IP₃R3. Practicable concentrations of caffeine and 2-APB inhibit only IP₃R1. Xestospongins do not appear to be effective antagonists of IP₃Rs.     

Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein

Molecular chaperones are an essential part of the machinery that avoids protein aggregation and misfolding in vivo. However, understanding the molecular basis of how chaperones prevent such undesirable interactions requires the conformational changes within substrate proteins to be probed during chaperone action. Here we use single-molecule fluorescence spectroscopy to investigate how the DnaJ–DnaK chaperone system alters the conformational distribution of the denatured substrate protein rhodanese. We find that in a first step the ATP-independent binding of DnaJ to denatured rhodanese results in a compact denatured ensemble of the substrate protein. The following ATP-dependent binding of multiple DnaK molecules, however, leads to a surprisingly large expansion of denatured rhodanese. Molecular simulations indicate that hard-core repulsion between the multiple DnaK molecules provides the underlying mechanism for disrupting even strong interactions within the substrate protein and preparing it for processing by downstream chaperone systems.Maintaining protein homeostasis in vivo requires a tight regulation of protein folding to prevent misfolding and aggregation. Molecular chaperones have evolved as an essential part of the cellular machinery that facilitates such processes in the complex and crowded environment of a living cell (1, 2). To assist protein folding, many chaperones proceed through complex conformational cycles in an ATP-dependent manner (3–5). For several chaperone systems, these cycles have been investigated in great detail by experiment and simulation (6–8). A remarkable example are the heat shock protein (Hsp) 70 chaperones, which are essential in prokaryotes and eukaryotes and are involved in co-translational folding, refolding of misfolded and aggregated proteins, protein translocation, and protein degradation (9). The Hsp70 chaperone DnaK from Escherichia coli together with its co-chaperone DnaJ and the nucleotide exchange factor GrpE form an ATP-driven catalytic reaction cycle (7) (Fig. 1A). Many denatured or misfolded substrate proteins are first captured by DnaJ and subsequently transferred to the DnaK–ATP complex, with DnaK in an open conformation. Substrate and DnaJ synergistically trigger DnaK’s ATPase activity, which leads to locking of the substrate in the DnaK–ADP complex, with DnaK in the closed conformation. Driven by the following GrpE-catalyzed ADP–ATP exchange, the DnaK–substrate complex dissociates (10). Since this ATP-driven cycle can even solubilize protein aggregates (11, 12), substantial forces must be transduced to the substrate protein (13–15). However, as for other chaperone systems (16), surprisingly little is known about how these forces and the resulting constraints of the underlying free energy surfaces affect the conformations of the denatured or misfolded substrate proteins. To better understand this important link between chaperone action and function, we probed the conformation of a substrate protein along the different stages of the chaperone cycle of DnaK with single-molecule Förster resonance energy transfer (smFRET), correlation spectroscopy, and microfluidic mixing.Open in a separate windowFig. 1.DnaK expands the denatured substrate protein. (A) Illustration of the DnaK–ATPase cycle. (B) Surface representation of rhodanese (PDB ID code 1RHS) with the subdomains indicated in different gray levels and the label positions of fluorescent dyes for single-molecule FRET measurements shown schematically. (C) FRET efficiency histograms of native rhodanese (gray) and denatured rhodanese under native conditions transiently populated in the microfluidic mixer (colored, measured 125 ms after dilution of rhodanese into native conditions). (D) FRET efficiency histograms of DnaJ–rhodanese complexes (0.5 µM DnaJ). (E) FRET efficiency histograms of DnaK–rhodanese complexes (0.5 µM DnaJ, 10 µM DnaK, and 1 mM ATP; DnaK and DnaJ were added simultaneously to rhodanese). Black lines indicate the DnaK–rhodanese complex population resulting from a fit that takes into account the residual population of refolded and DnaJ-bound rhodanese. The vertical lines in C–E indicate the positions of the FRET efficiency peaks of the native population of the respective rhodanese variants. The small populations at zero transfer efficiency in D (note the axis scaling and the small amplitudes of this population compared with E) originate from incomplete elimination of molecules with inactive acceptor fluorophores by pulsed interleaved excitation.     

Optimization of GPR40 Agonists for Type 2 Diabetes

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Diagnostic utility of urine cytology in detection of decoy cells in renal transplant patients: Report of five cases and review of literature

BK polyoma virus (PV) is one of the commonest post‐transplant viral infections, affecting approximately 15% of renal transplantation recipients, leading to graft failure in more than half of cases. The epithelial cells with polyoma viral inclusions in urine cytology specimens are termed “decoy cells” to caution pathologists not to misdiagnose these cells as cancer cells. The infected cells in urinary sediments are characterized by enlarged nucleus, basophilic intranuclear homogenous inclusions, and ground glass chromatin, which may cause diagnostic error in urine cytology. We report five cases of renal transplant patients, in which urine sample was positive for decoy cells. Routine urine cytology of post renal transplant patients with worsening renal function is a useful screening procedure to rule out PV reactivation, before ascertaining transplant rejection. Its cost‐effectiveness in addition to the short processing time makes it an invaluable tool in the evaluation of transplant recipients with symptoms suggestive of graft rejection.     

The role of intratumoral lymphovascular density in distinguishing primary from secondary mucinous ovarian tumors

OBJECTIVE:

Ovarian mucinous metastases commonly present as the first sign of the disease and are capable of simulating primary tumors. Our aim was to investigate the role of intratumoral lymphatic vascular density together with other surgical-pathological features in distinguishing primary from secondary mucinous ovarian tumors.

METHODS:

A total of 124 cases of mucinous tumors in the ovary (63 primary and 61 metastatic) were compared according to their clinicopathological features and immunohistochemical profiles. The intratumoral lymphatic vascular density was quantified by counting the number of vessels stained by the D2-40 antibody.

RESULTS:

Metastases occurred in older patients and were associated with a higher proportion of tumors smaller than 10.0 cm; bilaterality; extensive necrosis; extraovarian extension; increased expression of cytokeratin 20, CDX2, CA19.9 and MUC2; and decreased expression of cytokeratin 7, CA125 and MUC5AC. The lymphatic vascular density was increased among primary tumors. However, after multivariate analysis, the best predictors of a secondary tumor were a size of 10.0 cm or less, bilaterality and cytokeratin 7 negativity. Lack of MUC2 expression was an important factor excluding metastasis.

CONCLUSIONS:

The higher intratumoral lymphatic vascular density in primary tumors when compared with secondary lesions suggests differences in the microenvironment. However, considering the differential diagnosis, the best discriminator of a secondary tumor is the combination of tumor size, laterality and the pattern of expression of cytokeratin 7 and MUC2.     

p40 (ΔNp63) is more specific than p63 and cytokeratin 5 in identifying squamous cell carcinoma of bronchopulmonary origin: A review and comparative analysis

The therapeutic options now available for pulmonary squamous cell carcinoma (SQCC) and adenocarcinoma (ADC) are very different. The increasing demand to make a diagnosis on minimal tissue, ancillary techniques such as immunohistochemistry (IHC) are need to be highly sensitive and specific. The IHC marker p40 (ΔNp63) is a truncated isoform of p63 that is a promising IHC marker for SQCC. In this study, we have compared the specificity of p40(ΔNp63) IHC with p63 and cytokeratin 5 (CK5) on fine‐needle aspiration (FNA) cell blocks (CB). Thirty cases of pulmonary SQCC and 30 cases of pulmonary ADC with CB were selected. IHC for p40(ΔNp63), p63, and CK5 were performed on all paraffin‐embedded CB serial sections. All cases (n = 30) of SQCC stained positive for p40(ΔNp63). All cases of bronchopulmonary ADC were negative for both p40(ΔNp63) and CK5. Six cases (20%) of bronchopulmonary ADC demonstrated nuclear staining for p63 in at least 10% of malignant cells. Our data support p40(ΔNp63) to be more sensitive and specific and possess a greater positive and negative predictive value for SQCC in comparison to p63. This study also documents that p40(ΔNp63) does not stain ADC, which p63 does in 20% of the cases. We also found that p40(ΔNp63) shows a greater sensitivity and negative predictive value when compared to CK5. In paucicellular CB the increased indices p40(ΔNp63) provides may be extremely helpful in confirming the diagnosis of SQCC, which may have significant therapeutic implications. Diagn. Cytopathol. 2014;42:453–458. © 2013 Wiley Periodicals, Inc.     

CD40L基因c.674T>C突变的X连锁高IgM综合征1例报道

目的探讨高IgM综合征的特点、临床表现、实验室检查与治疗。方法回顾性分析1例X连锁高IgM综合征患者的临床资料。结果患儿以反复呼吸道感染入院,免疫功能检查提示IgG、IgA明显降低,IgM明显升高;基因检测结果示CD40L基因c.674T>C突变,给予抗感染、定期静脉滴注人免疫球蛋白等治疗。结论高IgM综合征临床表现缺乏特异性,依靠基因检测进行诊断;规律人免疫球蛋白替代治疗可能会提高患儿生存质量,延长寿命。