Early development of CD8-negative and CD8-positive MHC-unrestricted cytotoxic cells induced by alloantigen inoculation in mice

Peritoneal cells (PC) in C57B1/6 (B6, H-2b) mice receiving an intraperitoneal (i.p.) injection of allogeneic BALB/c (H-2d) spleen cells demonstrated potent cytotoxic activity against syngeneic, xenogeneic, third-party allogeneic tumors as well as H-2d derived tumors. Maximum cytotoxic activity against various tumors other than H-2d derived tumor, B16 (H-2b) was elicited on day 3 post allosensitization and decreased drastically thereafter, whereas cytotoxic activity against P815 (H-2d) peaked 3 days after the inoculation and maintained the peak activity thereafter. Surface phenotype of PC responsible for the cytotoxic activity against B16 was Thy-1+/-, Lyt-2-, L3T4-, asialo GM1 (AGM1)+, and that of PC against P815 was Thy-1+, Lyt-2+ (or Lyt-2+/-), L3T4-, AGM1+. These phenotypes showed similar phenotypes to the counterparts against B16 and against P815 in spleen cells induced by intravenous inoculation of alloantigen. When mice were pretreated i.p. with anti-AGM1 antibody before the allosensitization, anti-P815 cytotoxic-activity in PC was completely diminished. Similar activity in spleen, however, was enhanced by i.v. treatment with the antibody before the i.v. inoculation of alloantigen. The data clearly demonstrate that in vivo inoculation of B6 mice with normal allogeneic cells induces "NK-like" CD8- cytotoxic cells and "anomalous" CD8+ cytotoxic cells in PC.     

MHC-unrestricted recognition of bacteria-infected target cells by human CD8+ cytotoxic T lymphocytes.

A CD8+ alpha beta TCR+ T cell clone (A35) was isolated from the synovial fluid of a patient with post-enteric reactive arthritis caused by Yersinia enterocolitica. This clone efficiently killed autologous and allogeneic target cells that had been preincubated with live but not with heat-killed bacteria. There was no restriction by polymorphic parts of HLA-A, -B, or -C molecules and a HLA class II-deficient mutant cell line was lysed as efficiently as its normal counterpart, whereas infected HLA class I-deficient cells (Daudi cells) were not. The clone showed crossreaction between Yersinia enterocolitica, Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pyogenes, but did not lyse target cells preincubated with Staphylococcus epidermidis. MAb to CD2, CD3, and CD8 efficiently blocked A35, whereas the addition of mAb to HLA class II or to HLA class I did not. This clone apparently represents a novel effector mechanism against bacteria-infected or -modified cells that could be involved in the immunopathology of reactive arthritis.     

Adoptive transfer of CD8+ T cells from immune animals does not transfer immunity to blood stage Plasmodium yoelii malaria

The malaria parasite, Plasmodium yoelii 17X, causes a self-limited, nonlethal infection characterized, in the blood stage, by preferential invasion of reticulocytes. Previous studies have suggested that immunity to the blood stage infection may be related to enhanced levels of class I MHC Ag on the parasitized reticulocyte surface and can be adoptively transferred to immunodeficient mice by immune CD8+ T cells in the absence of CD4+ T cells. To further examine the mechanisms of CD8+ T cell involvement in immunity to blood stage P. yoelii infection, we performed in vivo CD8 depletion and adoptive transfer experiments. Depletion of CD8+ T cells during primary blood stage infection in BALB/c mice did not diminish the ability of the mice to resolve their infections. Spleen cells from immune BALB/c and C57BL/10 mice were transferred to BALB/c-nu/nu and C57BL/10-nu/nu mice, respectively. The recipient mice were CD4 depleted in vivo to kill any transferred CD4+ T cells. The mice failed to control the infection. Populations of CD4-, CD8+ T cells were transferred from immune CBA/CaJ donors to in vivo CD4-depleted CBA/CaJ recipients. The mice were unable to control the infection. Although immune unfractionated spleen cells transferred rapid protection in all three mouse strains and immune CD4+ T cells transferred immunity in the two mouse strains studied, CD8+ T cells by themselves were neither protective nor did they enhance immunity.     

Protective cellular retroviral immunity requires both CD4+ and CD8+ immune T cells.

We have found previously that postexposure chemoprophylaxis with 3'-azido-3'-deoxythymidine (also known as zidovudine or AZT) in combination with recombinant human alpha A/D interferon fully protected mice exposed to a lethal dose of Rauscher murine leukemia virus (RLV) against viremia and disease. After cessation of therapy, over 90% of these mice were able to resist rechallenge with live RLV, thus demonstrating an acquired immunity. Adoptive cell transfer of 4 x 10(7) cells from immunized mice fully protected naive recipients from viremia and splenomegaly after RLV challenge. However, when these immune T cells were fractionated into CD4+ and CD8+ subpopulations, only partial protection was found when 4 x 10(7) T cells of either subset were given. Full protection against RLV challenge was seen again when the T-cell subsets from immunized mice were recombined and transferred at the same number into naive mice. We conclude that cellular immunity alone is protective and that both CD4+ and CD8+ cell types are required for conferring full protection against live virus challenge.     

CD4-CD8- human T cells: phenotypic heterogeneity and activation requirements of freshly isolated "double-negative" T cells

In the present study we have analyzed the in vitro activation requirements of freshly isolated CD4-CD8- "double-negative" (DN) human peripheral blood T cells. DN cells were isolated from E+ cells by removal of CD4+, CD8+, and CD16+ cells through consecutive steps of C'-mediated lysis and panning. While the majority (79.0 +/- 12.0%) of DN cells were TCR gamma delta+ as shown by staining with mAb TCR delta-1, a minor fraction (6.7 +/- 4.7%) expressed TCR alpha beta as revealed by staining with mAb BMA031. Within the gamma delta+ DN fraction, most cells reacted with mAb Ti gamma A which delineates a V gamma 9JPC gamma 1 epitope, whereas a minor fraction stained with mAb delta TCS-1 which identifies a V delta 1J delta 1 epitope. Functional studies performed at low cell number (1000) per microculture indicated that DN cells can be activated by anti-CD3 mAb, PHA and allogeneic stimulator cells, provided that exogenous growth factors are supplied. Both rIl-2 and rIl-4 acted as efficient growth factors for DN cells, and a synergistic stimulatory effect of rIl-2 and rIl-4 was observed when DN cells were cocultured with allogeneic LCL stimulator cells. As compared to unseparated E+ cells, isolated DN responder cells had a reduced capacity to secrete Il-2 upon PHA stimulation in the presence of LCL feeder cells. The majority of DN cells maintained their CD3+ CD4-CD8- phenotype upon coculture with allogeneic LCL stimulator cells. These data demonstrate that CD3+ DN cells in human peripheral blood are heterogeneous with respect to TCR expression. In addition, they show that freshly isolated DN cells are deficient in Il-2 production but may be normally stimulated by anti-CD3, PHA, or alloantigen if exogenous growth factors (rIL-2 and/or rIl-4) are provided.     

Adoptive transfer of CD8alpha+ dendritic cells (DC) isolated from mice infected with Chlamydia muridarum are more potent in inducing protective immunity than CD8alpha- DC

Chlamydial infections are serious public health concerns worldwide. In this study, we examined the role of dendritic cell (DC) subsets in inducing protective immunity against chlamydial infection using an adoptive transfer approach. We found that CD11c+CD8alpha+ (double-positive, DP) DC, compared with CD11c+CD8alpha- (single-positive, SP) DC isolated from infected mice, are more potent inducers of protective immunity. Specifically, mice pretreated with DPDC from infected mice, upon infection with Chlamydia trachomatis mouse pneumonitis (MoPn), experienced significantly less severe body weight loss and in vivo chlamydial growth. Analysis of MoPn-driven cytokine production by immune cells revealed that mice that were treated with DPDC produced significantly higher levels of Th1 (TNF-alpha, IFN-gamma, and IL-12) but lower levels of Th2 (IL-4, IL-5, and IL-13)-related cytokines than the recipients of SPDC following infection challenge. Moreover, DPDC-treated mice displayed significantly higher levels of MoPn-specific IgG2a production and delayed-type hypersensitivity responses compared with SPDC-treated mice. Furthermore, DPDC isolated from infected mice produced higher amounts of IL-12 and IL-10 in vitro in comparison with SPDC. These data indicate that CD8alpha+ DC have a significantly higher capacity in inducing protective immunity compared with CD8alpha- DC, demonstrating the crucial role of DC1-like cells in eliciting protection against C. trachomatis infection.     

The new numerology of immunity mediated by virus-specific CD8(+) T cells

Our understanding of virus-specific CD8(+) T cell responses is currently being revolutionized by peptide-based assay systems that allow flow cytometric analysis of effector and memory cytotoxic T lymphocyte populations. These techniques are, for the first time, putting the analysis of T-cell-mediated immunity on a quantitative basis.     

Protection from lethal infection by adoptive transfer of CD8 T cells genetically engineered to express virus-specific innate immune receptor

CMV infection is one of the most common complications in immunocompromised individuals, such as organ and bone marrow transplant patients. Both innate and adaptive immune responses are required for defense against CMV infection. In murine CMV (MCMV) infection, strains harboring the MCMV-specific NK cell activation receptor, Ly49H (Klra8), are resistant. In contrast, MCMV infection of mice lacking Ly49H gene causes early mortality due to uncontrolled viral replication. In this study, we report the successful protection of mice from lethal MCMV infection with gene-transferred polyclonal CD8 T cells. CD8 T cells expressing a chimeric receptor comprising Ly49H extracellular and CD3zeta cytoplasmic domains are capable of killing target cells expressing the MCMV protein, m157. CD8 T cells expressing the chimeric receptor protect mice in vivo from lethality in the acute phase of MCMV infection, leading to the establishment of long-term protection. These data provide proof-of-principle evidence that a novel strategy for harnessing CD8 cytolytic function through TCR-independent yet pathogen-specific receptor can result in effective protection of hosts from pathogens.     

Passive transfer of tumor immunity with spleen cells from guinea pigs cured of hepatocarcinoma by nonspecific immunotherapy

Summary The purpose of this study was to evaluate cell-mediated tumor immunity in strain-2 guinea pigs cured of line-10 hepatocarcinoma by oil-in-water emulsions containing phenol-water extracts from either BCG or the Re mutant of Salmonella typhimurium (Re ET) admixed with mycobacteria glycolipid (P3). Treatment with these emulsions produced complete regression of established tumor nodules and prevented the growth of lymph node metastases in 25 of the 28 animals inoculated intradermally (ID) with 10⁶ line-10 cells and given intralesional immunotherapy 6 days later. No tumor regression was observed in animals given phenol-water extracts alone. Spleen cells, taken from guinea pigs cured of line-10 by BCG extract + P3 or Re ET + P3, were tested for their influence on tumor growth by means of an in vivo adoptive neutralization test (Winn test). Cell transfer was accomplished by the subcutanous injection of various concentrations of spleen cells admixed with 10⁵ viable line-10 cells. The results showed that as few as 10⁷ immune spleen cells completely inhibited the growth of 10⁵ tumor cells in 46–54% of the animals. The best tumor growth inhibition (77–85%) was observed in animals given 5 × 10⁷ immune cells admixed with 10⁵ tumor cells. The onset of transferrable tumor immunity was earlier in animals treated with the BCG extract + P3 than in those given the Re ET + P3. However, the duration of detectable tumor immunity was longer in the latter group. In contrast, no inhibition of tumor growth was observed in animals given spleen cells from normal or tumor-bearing guinea pigs. Moreover, spleen cells obtained from guinea pigs immunized with BCG extract + P3 or Re ET + P3 emulsions only and admixed with line-10 cells failed to transfer tumor immunity to normal animals. Thus, results from this study clearly demonstrated that cell-mediated tumor immunity was elicited in animals cured of line-10 tumor with combinations of P3 and phenol-water extracts of either BCG or Re mutant of S. typhimurium and that sensitized spleen cells effectively transferred systemic tumor immunity to normal recipients.     

Metabolic properties of freshly isolated bovine endothelial cells

Studies on endothelial cells metabolism have been limited by the lack of availability of a procedure for obtaining such cells in quantities adequate for direct in vitro analysis. Viable and well-dispersed endothelial cells in high yield have been obtained from the cavernous bodies of bovine penis. A preliminary characterization of the metabolic properties of the isolated cells has concerned the respiratory activity and some aspects of the carbohydrate metabolism. This study points out the following metabolic characteristics of endothelial cells: (1) a low respiration that is inhibited by high glucose concentrations (Crabtree effect); (2) a very high glycolytic activity in aerobic conditions, with a low Pasteur effect; (3) a very high potential activity of the hexose monophosphate shunt relative to the actual flux in the pathway. The biological relevance of these observations is discussed.     

Simultaneous induction of CD4 T cell tolerance and CD8 T cell immunity by semimature dendritic cells

Previous studies suggested that depending on their maturation state, dendritic cells (DC) could either induce T cell tolerance (immature and semimature DC) or T cell activation (mature DC). Pretreatment of C57BL/6 mice with encephalitogenic myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide-loaded semimature DC protected from MOG-induced autoimmune encephalomyelitis. This protection was mediated by IL-10-producing CD4 T cells specific for the self Ag. Here we show that semimature DC loaded with the MHC class II-restricted nonself peptide Ag (OVA) induce an identical regulatory T cell cytokine pattern. However, semimature DC loaded simultaneously with MHC class II- and MHC class I-restricted peptides, could efficiently initiate CD8 T cell responses leading to autoimmune diabetes in a TCR-transgenic adoptive transfer model. Double-peptide-loaded semimature DC also induced simultaneously in the same animal partially activated CD8 T cells with cytolytic function as well as protection from MOG-induced autoimmune encephalomyelitis. Our study suggests that the decision between tolerance and immunity not only depends on the DC, but also on the type and activation requirements of the responding T cell.     

Non-specific immune suppression by CD8+ T cells in Brugia pahangi-infected rats

Non-specific suppression of the immune response was investigated in Brugia pahangi-infected Lewis rats. The proliferative response of peripheral blood lymphocytes or splenic non-adherent cells to mitogens was significantly reduced by B. pahangi infection. The degree of hyporesponsiveness of splenic non-adherent cells to mitogens was comparable between microfilaremic and non-microfilaremic animals. The suppressed proliferative response of splenic non-adherent cells was restored by blocking with anti-CD8 monoclonal antibody. After separation of T cells into CD4+ and CD8+ subpopulations, only CD8+ T cells from B. pahangi-infected rats suppressed the proliferative response of normal spleen cells to concanavalin A. CD8+ T cells from normal rats had no suppressive effect. On the other hand, the proliferative response of CD4+ T cells to concanavalin A was comparable between normal and infected rats. These results suggest that CD8+ T cells participate in the non-specific suppression of immune response in experimental filariasis.     

Heterogeneity of freshly isolated human tonsil dendritic cells demonstrated by intracellular markers,phagocytosis, and membrane dye transfer

BACKGROUND: Heterogeneity within human dendritic cells (DCs) has been described but its functional relationships to cells of macrophage lineage and its role in human immunodeficiency virus (HIV) infection in vivo remain unclear. METHODS: Tonsil macrophages and DCs were isolated from low-density cells by negative selection and DCs were sorted into myeloid and plasmacytoid populations using antibodies to CD11c or CD123. Phagocytosis of latex beads and uptake of dye-labeled target cells were compared by flow cytometry and CD68 and S-100 by immunofluorescence on cytospins of sorted cells. RESULTS: Bead uptake and membrane dye transfer were found in both blood and tonsil CD11c(+) DCs and in CD14(+) cells particularly from blood monocytes. CD11c(-) DCs were poorly phagocytic but took up fluorescent dye from intact, necrotic or apoptotic cells. Tonsil DCs and macrophages expressed both CD68 and S-100 but CD11c(-) DCs expressed CD68 only. CONCLUSIONS: Freshly isolated CD11c(+) tonsil DCs are similar to CD14(+) macrophages in phagocytic function but the poorly phagocytic CD11c(-) DCs can also take up membrane from target cells. The intracellular markers commonly used to identify DCs and macrophages in situ do not identify accurately the CD11c(-) DC subset nor do they distinguish tonsil macrophages from DCs.     

Characterization of alpha beta+ CD4- CD8- CTL lines isolated from mixed lymphocyte cultures of adult mouse spleen cells.

Several CD4- CD8- and CD4- CD8+ T cell lines and clones have been isolated from the same mixed lymphocyte cultures. They express the alpha beta T cell receptor, are CD3+, V beta 8- and heat stable antigen-, and exhibit highly specific cytolytic activity mapping to H2Db. However, monoclonal antibody against H2Db failed to block lysis by CD4- CD8- cytotoxic T lymphocytes (CTL). One possible explanation is that the "double negative" cytotoxic T lymphocytes may recognize H2Db in the context of other class I molecules (Qa/Tla). CD4 and CD8 could be detected on some lines cloned in high concentrations of EL4 supernatant and the cell surface expression was influenced by growth conditions. The alpha beta+ CD4- CD8- CTL described in this report are clearly different from the so-called "autoimmune double negatives" which suggests a function for them in normal immune responses.     

Kinetics for the secretion of nonhelical procollagen by freshly isolated tendon cells

Fibroblasts isolated by enzymic digestion of chick embryo tendons have previously been used to examine the kinetics for the secretion of procollagen (Kao, W. W.-Y., Berg, R. A., and Prockop, D. J. (1977) J. Biol. Chem. 252, 8391-8397). The results indicated that the kinetics approximated the sum of two first order processes with half-times of 14 and 115 min. Here, the same fibroblasts were incubated in the presence of 1.53 mM cis-4-hydroxyproline, an analogue of proline, or in the presence of 0.3 mM alpha,alpha'-dipyridyl, an inhibitor of prolyl hydroxylase, so that the cells synthesized procollagen which could not assume a triple helical conformation characteristic of procollagen. Measurements of the secretion of nonhelical procollagen indicated that the kinetics for secretion differed from the kinetics for the secretion of procollagen and approximated a single first order process with a half-time of approximately 130 min. The nonhelical procollagen synthesized and secreted in the presence of either cis-4-hydroxyproline or alpha,alpha'-dipyridyl consisted of disulfide-bonded pro gamma chains of type I procollagen. The results suggested that the intracellular nonhelical procollagen was present in a single metabolic pool and secretion from this pool occurred with a different rate-limiting step than for helical procollagen. Further results indicated that nonhelical procollagen had a high affinity for prolyl hydroxylase and the affinity for the enzyme was greatly reduced if the procollagen was allowed to assume the triple helical conformation characteristic of normal procollagen. The results are consistent with the hypothesis that the secretion of procollagen is influenced by its conformation-dependent interaction with prolyl hydroxylase or other post-translational enzymes.     

Uptake of immune RNA by normal mouse spleen cells

Summary Normal mouse spleen cells take up in vitro radioactively labeled immune RNA. RNA taken up is present in nuclei, polysomes, membranes and cytoplasm. About 20–40% of immune RNA is nonspecifically associated with cell surface. 45% of RNA taken up is degraded and reutilized inside the cells within 2 hours.This work was supported by the Polish Academy of Sciences within the project 09.7.4.1.1.