Hirohito Kobayashi

Monokine Induced by IFN-γ Is a Dominant Factor Directing T Cells into Murine Cardiac Allografts During Acute Rejection

The use of chemokine antagonism as a strategy to inhibit leukocyte trafficking into inflammatory sites requires identification of the dominant chemokines mediating recruitment. The chemokine(s) directing T cells into cardiac allografts during acute rejection remain(s) unidentified. The role of the CXC chemokines IFN-γ inducible protein 10 (IP-10) and monokine induced by IFN-γ (Mig) in acute rejection of A/J (H-2a) cardiac grafts by C57BL/6 (H-2b) recipients was tested. Intra-allograft expression of Mig was observed at day 2 posttransplant and increased to the time of rejection at day 7 posttransplant. IP-10 mRNA and protein production were 2.5- to 8-fold lower than Mig. Whereas allografts were rejected at day 7–9 in control recipients, treatment with rabbit antiserum to Mig, but not to IP-10, prolonged allograft survival up to day 19 posttransplant. At day 7 posttransplant, allografts from Mig antiserum-treated recipients had marked reduction in T cell infiltration. At the time of rejection in Mig antiserum-treated recipients (i.e., days 17–19), intra-allograft expression of macrophage-inflammatory protein-1α, -1β, and their ligand CCR5 was high, whereas expression of CXCR3, the Mig receptor, was virtually absent. Mig was produced by the allograft endothelium as well as by recipient allograft-infiltrating macrophages and neutrophils, indicating the synergistic interactions between innate and adaptive immune compartments during acute rejection. Collectively, these results indicate that Mig is a dominant recruiting factor for alloantigen-primed T cells into cardiac allografts during acute rejection. Although Mig antagonism delays acute heart allograft rejection, the results also suggest that the alloimmune response circumvents Mig antagonism through alternative mechanisms.

Gamma/delta T cells provide innate immunity against renal cell carcinoma.

Abstract Host immune function plays a certain role against the development of renal cell carcinomas (RCCs), but the mechanism is not entirely understood. Human gamma/delta (γ/δ) T cells defend the body against infection. In this study, we clarify the role of γ/δ T cells in the surveillance system against RCCs by analyzing the γ/δ T cells in peripheral blood mononucleocytes (PBMs) and tumor infiltrating lymphocytes (TILs) from 41 patients with RCCs. The results showed that the number of γ/δ T cells expressing Vγ2 and Vδ2 in variable elements of TCR was elevated in the PBMs in 10 patients, but not in any of 32 healthy individuals. The proportion of patients with an elevated number of γ/δ T cells (>10%) increased with cancer stage. The level of the γ/δ T cells decreased after surgery. The γ/δ T cells in the TILs were more activated than those in the PBMs. Evaluation of the junctional diversity of TCR Vγ2 and Vδ2 chains showed that the increased peripheral blood γ/δ T cells were oligoclonal rather than polyclonal. Taken together, our findings suggest that γ/δ T cells recognize certain RCC-related antigens and play a role in the surveillance system against RCCs.

Template-based lymphadenectomy in urothelial carcinoma of the upper urinary tract: Impact on patient survival

Objectives:  The benefit of lymphadenectomy (LND) in patients with urothelial carcinoma of the upper urinary tract (UCUUT) has remained controversial. The aim of this study was to examine the influence of the LND template and the total number of lymph nodes (LN) when increasing the number of patients undergoing complete dissection of regional nodes (CompLND).

Expression and function of PD-1 in human γδ T cells that recognize phosphoantigens

Programmed cell death‐1 (PD‐1) is an inhibitory receptor and plays an important role in the regulation of αβ T cells. Little is known, however, about the role of PD‐1 in γδ T cells. In this study, we investigated the expression and function of PD‐1 in human γδ T cells. Expression of PD‐1 was rapidly induced in primary γδ T cells following antigenic stimulation, and the PD‐1+ γδ T cells produced IL‐2. When PD‐1+ γδ T cells were stimulated with Daudi cells with and without programmed cell death ligand‐1 (PD‐L1) expression, the levels of IFN‐γ production and cytotoxicity in response to PD‐L1+ Daudi cells were diminished compared to the levels seen in response to PD‐L1− Daudi cells. The attenuated effector functions were reversed by anti‐PD‐L1 mAb. When PD‐1+ γδ T cells were challenged by PD‐L1+ tumors pretreated with zoledronate (Zol), which induced γδ TCR‐mediated signaling, the resulting reduction in cytokine production was only slight to moderate compared to the reduction seen when PD‐1+ γδ T cells were challenged by PD‐L1− tumors. In addition, cytotoxic activity of PD‐1+ γδ T cells against Zol‐treated PD‐L1+ tumors was comparable to that against Zol‐treated PD‐L1− tumors. These results suggest that TCR triggering may partially overcome the inhibitory effect of PD‐1 in γδ T cells.

Template-based lymphadenectomy in urothelial carcinoma of the renal pelvis: A prospective study

Recent studies showed the therapeutic benefit of lymphadenectomy in advanced stage urothelial carcinoma of the upper urinary tract, but there is still a lack of prospective studies and standardization of the extent of lymphadenectomy. The aim of this multi‐institutional study was to examine the role of lymphadenectomy in urothelial carcinoma of the upper urinary tract.

Superior Tolerability of Altered Dosing Schedule of Sunitinib with 2-Weeks-on and 1-Week-off in Patients with Metastatic Renal Cell Carcinoma—Comparison to Standard Dosing Schedule of 4-Weeks-on and 2-Weeks-off

OBJECTIVE Poor tolerability to sunitinib with the standard dosing schedule has become an issue. We retrospectively analyzed the treatment efficacy and the profile of adverse events of 2 weeks of sunitinib treatment followed by 1-week-off (Schedule 2/1) and compared the results with the standard dosing schedule with 4 weeks of treatment followed by 2-weeks-off (Schedule 4/2). METHODS From January 2010 until December 2012, 48 patients with metastatic renal cell carcinoma who received at least two cycles of sunitinib as first-line therapy were the subjects of this study. After 2011, we switched to Schedule 2/1 for most patients. RESULTS Schedule 2/1 included 26 patients and Schedule 4/2 had 22. The incidence of most adverse events was not significantly different between the two groups except for hand-foot syndrome and diarrhoea, which were observed more frequently in Schedule 4/2 and reached statistical significance. A dose interruption due to adverse events in the first three cycles was significantly lower in Schedule 2/1 patients than in those on Schedule 4/2 (27 versus 53% P = 0.04). With respect to treatment efficacy, the objective response rate tended to be higher in Schedule 4/2 than in Schedule 2/1 (50 versus 32%), and median progression-free survival was longer in patients on Schedule 2/1 than those on Schedule 4/2 (18.4 versus 9.1 months). These differences, however, did not reach statistical significance (P = 0.14, P = 0.13). CONCLUSIONS Alteration in dosing schedule of sunitinib with 2-weeks-on and 1-week-off showed a lower incidence of dose interruption and a similar oncological outcome compared with the standard dosing schedule of 4-weeks-on and 2-weeks-off.

CXC Chemokine Ligand 9/Monokine Induced by IFN-γ Production by Tumor Cells Is Critical for T Cell-Mediated Suppression of Cutaneous Tumors

The role of tumor-produced chemokines in the growth of malignancies remains poorly understood. We retrieved an in vivo growing MCA205 fibrosarcoma and isolated tumor cell clones that produce both CXCL9/monokine induced by IFN-γ (Mig) and CXCL10/IFN-γ-inducible protein 10 following stimulation with IFN-γ and clones that produce IFN-γ-inducible protein 10 but not Mig. The Mig-deficient variants grew more aggressively as cutaneous tumors in wild-type mice than the Mig-producing tumor cells. The growth of Mig-expressing, but not Mig-deficient, tumor cells was suppressed by NK and T cell activity. Transduction of Mig-negative variants to generate constitutive tumor cell production of Mig resulted in T cell-dependent rejection of the tumors and in induction of protective tumor-specific CD8+ T cell responses to Mig-deficient tumors. The results indicate a critical role for tumor-derived Mig in T cell-mediated responses to cutaneous fibrosarcomas and suggest the loss of Mig expression as a mechanism used by tumor cells to evade these responses.

T-cell mediated induction of allogeneic endothelial cell chemokine expression.

Background. The goal of the current study was to test the ability of T cells to stimulate allogeneic endothelial cells to express chemokines, particularly the T-cell recruiting factors monokine induced by interferon-&ggr; (Mig) and inducible protein (IP)-10. Methods. Lymph node cells from C57BL/6 (H-2b) recipients of C3H (H-2k) skin grafts or from naïve mice were added to monolayers of C3H-derived endothelial cell line 2F-2B. After 5 or 24 hr, the lymph node cells were removed, and RNA was prepared from the endothelial cells and tested by ribonuclease protection assay or Northern blot hybridization for endothelial cell expression of chemokines. Results. Alloantigen-primed T cells induced endothelial cell expression of regulated on activation normal T-cell expressed and secreted (RANTES), IP-10, Mig, monocyte chemotactic protein-1, macrophage inflammatory protein-1&agr;, and macrophage inflammatory protein-1&bgr; within 5 hr of coculture. In vitro chemotaxis assays demonstrated the production of T-cell chemoattractants by the endothelial cells. With the exception of low levels of monocyte chemotactic protein-1 and RANTES, culture with naïve C57BL/6 lymph node T cells did not induce endothelial cell chemokine expression. Alloantigen-primed CD4+ T cells induced endothelial expression of IP-10 and RANTES but none of the other chemokines tested, whereas primed CD8+ T cells induced all of the chemokines tested. Expression of IP-10 and Mig was not induced when alloantigen-primed T cells from interferon-&ggr; deficient recipients of C3H skin grafts were cultured with the endothelial cells. This expression was blocked by addition of intercellular adhesion molecule-1 or lymphocyte function-associated antigen-1 specific antibodies to the cultures. Conclusions. These results demonstrate the ability of alloantigen-primed CD8+ T cells to quickly and directly stimulate endothelial cells to express and produce chemokines, including those recruiting T cells.

Chronic antagonism of Mig inhibits cellular infiltration and promotes survival of class II MHC disparate skin allografts.

Background. The goal of the current study was to test the ability of monokine induced by IFN-&ggr; (Mig)-specific antibodies to inhibit long-term T cell infiltration into class II major histocompatibility complex (MHC) disparate skin allografts and to test cellular and molecular changes in the graft during the rejection observed following cessation of treatment. Methods. C57BL/6 recipients of B6.H-2bm12 skin grafts were treated with normal rabbit serum (NRS) or rabbit Mig antiserum (Mig AS) every other day from day 7 until day 21 posttransplant and then weekly thereafter. Allografts were retrieved during the course of treatment and following cessation. Tissue sections were prepared and stained to compare infiltration by macrophages and CD4+ and CD8+ T cells and to assess collagen deposition in the grafts. RNA was prepared and tested by ribonuclease protection assay for intragraft levels of Mig, monocyte chemotactic protein-1 (MCP-1) and regulated on activation normal T-cell expressed and secreted (RANTES). Results. T cell and macrophage infiltration into allografts was inhibited and graft survival maintained as long as Mig-specific antibodies were given. Following cessation of treatment, T cells and macrophages infiltrated the allografts. In contrast to the histology of acute rejection observed in allografts from NRS-treated recipients, the resulting rejection of the allografts from Mig AS-treated recipients was accompanied by dense collagen deposition and high level expression of Mig and RANTES. Conclusions. Mig directs T cell infiltration into B6.H-2bm12 skin allografts on C57BL/6 recipients. Delayed T cell and macrophage infiltration and rejection of the grafts following cessation of Mig AS treatment results in rejection that is histologically and molecularly distinct from acute rejection.

Presurgical Targeted Therapy with Tyrosine Kinase Inhibitors for Advanced Renal Cell Carcinoma: Clinical Results and Histopathological Therapeutic Effects

OBJECTIVES We retrospectively analyzed our patients with advanced renal cell carcinoma who underwent presurgical targeted therapy with tyrosine kinase inhibitors to clarify the safety and clinical benefit. The histopathological effect of this treatment was also examined. METHODS Between July 2005 and February 2010, nine patients with advanced renal cell carcinoma who were treated with tyrosine kinase inhibitors before surgery were the subjects of this study. Consolidative surgery was considered when these tumors showed clinical response or stable disease while on targeted therapy without evidence of disease progression at other sites. RESULTS The agents used were sorafenib in seven patients and sunitinib in two. The median duration of presurgical therapy was 12.2 weeks, and seven patients had less than 4 months of treatment. Tumor reduction at 10-30% was obtained in all patients but one. Perioperative complications were observed in five of nine patients. Major complications occurred in two patients, including intraoperative excessive bleeding and delayed localized intraperitoneal abscess. Minor complications were found in three. The characteristics of the histopathological effect of tyrosine kinase inhibitors consisted of marked atrophy of the capillary sinus, confirming the pharmacological mechanisms of these agents. Other findings included nuclear pyknosis and degeneration of tumor cells. CONCLUSIONS Presurgical targeted therapy with tyrosine kinase inhibitors appears to be feasible in most patients with advanced renal cell carcinoma. However, the indications, the clinical benefit and the standard protocol still remain to be determined. Therapeutic effects in the histology were compatible to their pharmacological effects.