Journal of Experimental Medicine

PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor–mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon γ, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.

CD4+CD25+ regulatory T cells (Treg) are instrumental in the maintenance of immunological tolerance. One critical question is whether Treg can only be generated in the thymus or can differentiate from peripheral CD4+CD25− naive T cells. In this paper, we present novel evidence that conversion of naive peripheral CD4+CD25− T cells into anergic/suppressor cells that are CD25+, CD45RB−/low and intracellular CTLA-4+ can be achieved through costimulation with T cell receptors (TCRs) and transforming growth factor β (TGF-β). Although transcription factor Foxp3 has been shown recently to be associated with the development of Treg, the physiological inducers for Foxp3 gene expression remain a mystery. TGF-β induced Foxp3 gene expression in TCR-challenged CD4+CD25− naive T cells, which mediated their transition toward a regulatory T cell phenotype with potent immunosuppressive potential. These converted anergic/suppressor cells are not only unresponsive to TCR stimulation and produce neither T helper cell 1 nor T helper cell 2 cytokines but they also express TGF-β and inhibit normal T cell proliferation in vitro. More importantly, in an ovalbumin peptide TCR transgenic adoptive transfer model, TGF-β–converted transgenic CD4+CD25+ suppressor cells proliferated in response to immunization and inhibited antigen-specific naive CD4+ T cell expansion in vivo. Finally, in a murine asthma model, coadministration of these TGF-β–induced suppressor T cells prevented house dust mite–induced allergic pathogenesis in lungs.

Interleukin (IL)-23 is a heterodimeric cytokine composed of a unique p19 subunit, and a common p40 subunit shared with IL-12. IL-12 is important for the development of T helper (Th)1 cells that are essential for host defense and tumor suppression. In contrast, IL-23 does not promote the development of interferon-γ–producing Th1 cells, but is one of the essential factors required for the expansion of a pathogenic CD4+ T cell population, which is characterized by the production of IL-17, IL-17F, IL-6, and tumor necrosis factor. Gene expression analysis of IL-23–driven autoreactive T cells identified a unique expression pattern of proinflammatory cytokines and other novel factors, distinguishing them from IL-12–driven T cells. Using passive transfer studies, we confirm that these IL-23–dependent CD4+ T cells are highly pathogenic and essential for the establishment of organ-specific inflammation associated with central nervous system autoimmunity.

Plaques have been produced with the three types of poliomyelitis viruses on monolayer tissue cultures of monkey kidney and monkey testis. The number of plaques was proportional to the concentration of the virus. Each plaque originates, therefore, from a single virus particle, defined as the virus unit that is unseparable by dilution. The plaques are due to the specific action of the virus since they are suppressed by type-specific antiserum.

Pure virus lines were established by isolating the virus population produced in single plaques. These derived virus lines had the same morphological, serological, and pathogenic properties as the parent strain.

High titer virus stocks, with titers up to 7 x 108 plaque-forming particles per ml., were obtained.

Nghiên cứu hiện tại chứng minh rằng bạch cầu đơn nhân người được kích hoạt bằng lipopolysaccharides (LPS) có khả năng sản xuất mức cao interleukin 10 (IL-10), trước đây được gọi là yếu tố ức chế tổng hợp cytokine (CSIF), phụ thuộc vào liều lượng. IL-10 có thể được phát hiện 7 giờ sau khi kích hoạt bạch cầu đơn nhân và mức tối đa của sự sản xuất IL-10 được quan sát sau 24-48 giờ. Những động học này chỉ ra rằng việc sản xuất IL-10 bởi bạch cầu đơn nhân người tương đối muộn so với sự sản xuất IL-1 alpha, IL-1 beta, IL-6, IL-8, yếu tố hoại tử khối u alpha (TNF alpha), và yếu tố kích thích thuộc địa bạch cầu trung tính (G-CSF), tất cả đều được tiết ra ở mức cao từ 4-8 giờ sau khi kích hoạt. Việc sản xuất IL-10 bởi bạch cầu đơn nhân được kích hoạt bởi LPS, tương tự như của IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, yếu tố kích thích thuộc địa bạch cầu đại thực bào (GM-CSF), và G-CSF, bị ức chế bởi IL-4. Hơn nữa, chúng tôi chứng minh rằng IL-10, được thêm vào bạch cầu đơn nhân, khi được kích hoạt bởi interferon gamma (IFN-gamma), LPS, hoặc các tổ hợp của LPS và IFN-gamma vào đầu giai đoạn nuôi cấy, giảm mạnh sản xuất IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, và G-CSF ở mức phiên mã. Viral-IL-10, với các hoạt động sinh học tương tự trên tế bào người, cũng ức chế sản xuất TNF alpha và GM-CSF bởi bạch cầu đơn nhân sau khi kích hoạt LPS. Kích hoạt bạch cầu đơn nhân bằng LPS với sự hiện diện của các kháng thể đơn dòng trung hòa anti-IL-10 dẫn đến sản xuất một lượng cytokine lớn hơn so với điều trị chỉ với LPS, chỉ ra rằng IL-10 được sản xuất nội sinh đã ức chế sản xuất IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, và G-CSF. Ngoài ra, IL-10 có tác động tự điều hòa vì nó ức chế mạnh mẽ sự tổng hợp mRNA IL-10 trong bạch cầu đơn nhân được kích hoạt bằng LPS. Hơn nữa, IL-10 được sản xuất nội sinh được tìm thấy là chịu trách nhiệm cho việc giảm biểu hiện phức hợp hòa hợp mô chính II (MHC) sau khi bạch cầu đơn nhân được kích hoạt với LPS. Tóm lại, kết quả của chúng tôi chỉ ra rằng IL-10 có tác động điều hòa quan trọng trên các đáp ứng miễn dịch và viêm nhiễm do khả năng của nó làm giảm biểu hiện phức hợp MHC II và ức chế sản xuất các cytokine gây viêm bởi bạch cầu đơn nhân.

A critical event during programmed cell death (PCD) appears to be the acquisition of plasma membrane (PM) changes that allows phagocytes to recognize and engulf these cells before they rupture. The majority of PCD seen in higher organisms exhibits strikingly similar morphological features, and this form of PCD has been termed apoptosis. The nature of the PM changes that occur on apoptotic cells remains poorly defined. In this study, we have used a phosphatidylserine (PS)-binding protein (annexin V) as a specific probe to detect redistribution of this phospholipid, which is normally confined to the inner PM leaflet, during apoptosis. Here we show that PS externalization is an early and widespread event during apoptosis of a variety of murine and human cell types, regardless of the initiating stimulus, and precedes several other events normally associated with this mode of cell death. We also report that, under conditions in which the morphological features of apoptosis were prevented (macromolecular synthesis inhibition, overexpression of Bcl-2 or Abl), the appearance of PS on the external leaflet of the PM was similarly prevented. These data are compatible with the notion that activation of an inside-outside PS translocase is an early and widespread event during apoptosis.

By using the two criteria (a) high density of immunoglobulin determinants on the cell surface and (b) presence of receptors for C'3 on the cell surface for defining bone marrow-derived lymphocytes, it is indirectly shown that all or at least a major population of human thymus-derived lymphocytes under certain conditions will form nonimmune rosettes with sheep red blood cells (SRBC). Almost all thymocytes tested from two different donors formed rosettes. The SRBC rosettes are not formed by virtue of immunoglobulin receptors and form only around living cells. Positive bivalent ions are required for rosette formation since EDTA will block rosette formation. Sodium iodoacetate will also block rosette formation demonstrating the dependence on an intact glycolytic pathway. Rosette formation is temperature dependent and will not appear at 37°C. Trypsin treatment of lymphocytes will abolish their SRBC-binding ability which cannot be restored by treating them with fresh donor serum or fetal calf serum, but which will reappear after culturing the lymphocytes. It is suggested that these rosettes are formed by a rapidly released or metabolized receptor substance on the living cell surface which behaves as a trypsin-sensitive structure produced by the cells themselves.

Two methods were used to demonstrate the presence of tumor-specific antigens in adenocarcinomata of the human colon: (a) rabbits were immunized with extracts of pooled colonic carcinomata, and the antitumor antisera thus produced were absorbed with a pooled extract of normal human colon and with human blood components; (b) newborn rabbits were made immunologically tolerant to normal colonic tissue at birth, and were then immunized with pooled tumor material in adult life. Normal and tumor tissues were obtained from the same human donors in order to avoid misinterpretation of results due to individual-specific antigenic differences.

The antisera prepared by both methods were tested against normal and tumor antigens by the techniques of agar gel diffusion, immunoelectrophoresis, hemagglutination, PCA, and immunofluorescence. Distinct antibody activity directed against at least two qualitatively tumor-specific antigens, or antigenic determinants, was detected in the antisera prepared by both methods and at least two additional tumor antigens were detected exclusively in antisera prepared by the tolerance technique. Whether these additional antigens were qualitatively different from normal tissue antigens, or merely present in tumor tissue in higher concentrations than in normal tissue has not as yet been determined. Furthermore, it was shown that the tumor-specific antibodies were not directed against bacterial contaminants or against the unusually high concentrations of fibrin found in many neoplastic tissues.

It was concluded from these results that the pooled tumor extracts contained tumor-specific antigens not present in normal colonic tissue. Identical tumor-specific antigens were also demonstrated in a number of individual colonic carcinomata obtained from different human donors.

Activation in lectin-free interleukin 2 (IL-2) containing supernatants of peripheral blood mononuclear leukocytes (PBL) from cancer patients or normal individuals resulted in expression of cytotoxicity toward 20 of 21 natural killer (NK)-resistant fresh solid tumor cells tested. Fresh solid tumor cells were resistant to NK-mediated lysis in 10 autologous patients' PBL-tumor interactions, and from 17 normal individuals tested against 13 allogeneic fresh tumors. Culture of PBL in IL-2 for 2-3 d was required for the lymphokine activated killers (LAK) to be expressed, and lytic activity toward a variety of NK-resistant fresh and cultured tumor targets developed in parallel. Autologous IL-2 was functional in LAK activation, as well as interferon-depleted IL-2 preparations. Irradiation of responder PBL before culture in IL-2 prevented LAK development. Precursors of LAK were present in PBL depleted of adherent cells and in NK-void thoracic duct lymphocytes, suggesting that the precursor is neither a monocyte nor an NK cell. LAK effectors expressed the serologically defined T cell markers of OKT.3, Leu-1, and 4F2, but did not express the monocyte/NK marker OKM-1. Lysis of autologous fresh solid tumors by LAK from cancer patients' PBL was demonstrated in 85% of the patient-fresh tumor combinations. Our data present evidence that the LAK system is a phenomenon distinct from either NK or CTL systems that probably accounts for a large number of reported nonclassical cytotoxicities. The biological role of LAK cells is not yet known, although it is suggested that these cells may be functional in immune surveillance against human solid tumors.

It is now clear that functionally specialized regulatory T (Treg) cells exist as part of the normal immune repertoire, preventing the development of pathogenic responses to both self- and intestinal antigens. Here, we report that the Treg cells that control intestinal inflammation express the same phenotype (CD25+CD45RBlowCD4+) as those that control autoimmunity. Previous studies have failed to identify how CD25+ Treg cells function in vivo. Our studies reveal that the immune-suppressive function of these cells in vivo is dependent on signaling via the negative regulator of T cell activation cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), as well as secretion of the immune-suppressive cytokine transforming growth factor β. Strikingly, constitutive expression of CTLA-4 among CD4+ cells was restricted primarily to Treg cells, suggesting that CTLA-4 expression by these cells is involved in their immune-suppressive function. These findings raise the possibility that Treg cell function contributes to the immune suppression characteristic of CTLA-4 signaling. Identification of costimulatory molecules involved in the function of Treg cells may facilitate further characterization of these cells and development of new therapeutic strategies for the treatment of inflammatory diseases.