Production of specific macrophage-arming factor precedes cytotoxic T lymphocyte activity in vivo during tumor rejection

Summary Recently we published a hypothesis on the immunological events occurring during tumor rejection. One of the implications of this hypothesis is that specific macrophage-arming factor (SMAF) is produced early during the initiation of the immune response, whereas the classical cell-mediated immune response components, such as cytotoxic T lymphocytes (CTL), are produced later, that is, during the amplifier-effector phase. In this paper we establish the kinetics of the induction of (a) lymphocytes producing SMAF and (b) CTL. Groups of DBA/2 mice were injected i.p. once, twice or three times with irradiated and/or non-irradiated syngeneic SL2 tumor cells, the injections being given at intervals of 10 days. After each of these injections the production of SMAF and the expression of CTL activity were established. The results showed that in the peritoneal cavity SMAF-producing lymphocytes appeared earlier than cytotoxic lymphocytes (CTL). In addition, it was shown (a) that SMAF does not interfere with the in vitro cytotoxicity expressed by CTL and (b) that in addition to CTL memory cells, SMAF-producing memory cells were also induced after injection of syngeneic tumor cells. These data support the hypothesis that SMAF is involved in the early phase of the cellular immune response against tumors, whereas CTL are induced later.     

Involvement of lymphocyte mediators in the rejection of a murine tumor allograft

Macrophage migration inhibition by peritoneal leukocytes was studied in BALB/c mice bearing intraperitoneal allogeneic EL-4 lymphomas to explore the role of this immune effector function in allograft rejection. The nonadherent peritoneal leukocyte population harvested between 8 and 10 days after allograft inoculation inhibited migration of nonimmune murine macrophages as demonstrated by both direct and indirect migration assays using the agarose droplet method. This host response also contained large numbers of adherent macrophages which others have shown to be cytotoxic to EL-4 target cells. These findings provide direct evidence for lymphokine activity in allograft rejection and suggest that lymphocyte mediators may attract and activate the cytotoxic macrophages observed in this response.     

The immunological network at the site of tumor rejection

The tumor mass irrespective of its type or location in the body has long been shrouded in mystery and even today we still have only a tentative handle on its secrets. Attempts to manipulate either the tumor cells per se or host-derived leukocytes have, on the whole, not been successful or at best questionable. The ability of the host to respond immunologically to TSTA is well documented, yet again attempts to manipulate this response have been disappointing. One of the problems has been a lack of knowledge concerning the tumor mass and its constituents, such as the intratumor leukocytes, and the significance of their presence to the biological properties of the neoplasm [8,9,80]. The purpose in studying the immunological network is, in part, to try to assign a function to these cells on the premise that lymphoid elements and macrophages have a potential role to play in recognition of TSTA. The advantage of adoptive immunotherapy model systems is that tumor rejection can be achieved under controlled conditions and this allows an analysis of the immunological network and its individual circuits. At the same time, valuable information on the mechanisms of action during adoptive immunotherapy and how best to improve therapeutic protocols is acquired.     

Regulation and function of an insulin-sensitive glycosyl-phosphatidylinositol during T lymphocyte activation

A combination of metabolic labeling and chemical or enzymatic modification was employed to isolate and biochemically characterize a set of glycosyl-phosphatidylinositol (gly-PI) molecules synthesized by T lymphocytes. Gly-PI displayed unique patterns of synthesis following mitogen activation relative to the phosphoinositides and major structural lipids. The increase with time in gly-PI was paralleled by the appearance of insulin receptors. Gly-PI molecules were sensitive to hydrolysis by a PI-specific phospholipase C and were rapidly (15 sec) degraded in response to insulin binding. The product of this hydrolysis is believed to be a novel inositol phosphate-glycan (IP-gly) that was shown to inhibit the activity of a cAMP-dependent protein kinase. These results demonstrate that T cells contain a structurally related set of gly-PI molecules, at least one of which is sensitive to insulin and may function as a second messenger of hormone action.     

长丝萝中的两个抗肿瘤活性成分的分离鉴定及对抑癌基因变化的验证

以长丝萝Dolichousnea longissima中分离得到的体外细胞毒活性较强的两个苯骈呋喃类化合物为实验材料,采用Affymetrix全表达谱基因芯片、GO分类、Pathway分析和实时荧光定量PCR技术对2种化合物处理前后的肝癌细胞株HepG2的差异表达基因进行分析和验证。结果表明,(Z)-2-乙酰基-5,5-二[2-(7-乙酰基-4,6-二羟基-3,5-二甲基苯骈呋喃基)]-4-羟基-2,4-戊二烯-1-醛筛选出明显变化的基因728个,其中上调基因有246个,下调基因有482个。PATHWAY分析与肿瘤相关的信号通路有细胞周期信号通路、内吞作用信号通路、癌细胞中信号通路、前列腺癌信号通路、p53信号通路、结肠直肠癌信号通路。GO分类显示差异基因共参与266个BP分类、68个CC分类和43个MF分类。4-[3-(7-乙酰基-4,6-二羟基-3,5-二甲基-2-氧代-2,3-二氢苯骈呋喃基)]-4-[2-(7-乙酰基-4,6-二羟基-3,5-二甲基苯骈呋喃基)]-3-氧代丁酸乙酯筛选出明显变化的基因112个,其中上调基因有8个,下调基因有104个。PATHWAY分析与肿瘤相关的信号通路有细胞周期信号通路、内吞作用信号通路。GO分类显示差异基因共参与109个BP分类、48个CC分类和15个MF分类。因此,以上两个苯骈呋喃类化合物均主要通过影响细胞周期信号通路和内吞作用信号通路使抑癌基因发生变化。     

Idiotype network components are involved in the murine immune response to simian virus 40 large tumor antigen

Summary Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag), a monoclonal antibody specific for SV40 T-Ag (Ab-1 preparation), and a monoclonal anti-idiotypic antibody (anti-Id), designated 58D, were used to analyze the humoral immune response of Balb/c mice either immunized with recombinant SV40 T-Ag or challenged with SV40-transformed cells. Inhibition assays indicated that antibodies from mice immunized with SV40 T-Ag and from those bearing SV40 tumor inhibited the SV40 T-Ag/Ab-1 reaction. These data suggested that the antibody response in immunized or tumorchallenged mice recognized similar epitope(s) on SV40 T-Ag to that detected by the monoclonal Ab-1. These anti-(SV40 T-Ag) response antibodies also inhibited the Ab-1/anti-Id reaction and recognized the anti-Id in direct binding assays. Together, these data indicate that murine anti-(SV40 T-Ag) responses shared an idiotope with a monoclonal anti-(SV40 T-Ag) Ab-1 preparation. This idiotope, which is recognized by the monoclonal anti-Id preparation, 58D, appears to be involved in the humoral immune response to SV40 T-Ag in both SV40-T-Ag-immunized and tumor-bearing mice. The monoclonal anti-Id preparation may represent a focal point for manipulating the humoral immune response to tumors induced by SV40-transformed cells.     

Macrophage T lymphocyte interactions in the anti-tumor immune response: a mathematical model

In this paper we present a model of the macrophage T lymphocyte interactions that generate an anti-tumor immune response. The model specifies i) induction of cytotoxic T lymphocytes, ii) antigen presentation by macrophages, which leads to iii) activation of helper T cells, and iv) production of lymphoid factors, which induce a) cytotoxic macrophages, b) T lymphocyte proliferation, and c) an inflammation reaction. Tumor escape mechanisms (suppression, antigenic heterogeneity) have been deliberately omitted from the model. This research combines hitherto unrelated or even contradictory data within the range of behavior of one model. In the model behavior, helper T cells play a crucial role: Tumors that differ minimally in antigenicity (i.e., helper reactivity) can differ markedly in rejectability. Immunization yields protection against tumor doses that would otherwise be lethal, because it increases the number of helper T cells. The magnitude of the cytotoxic effector cell response depends on the time at which helper T cells become activated: early helper activity steeply increases the magnitude of the immune response. The type of cytotoxic effector cells that eradicates the tumor depends on tumor antigenicity: lowly antigenic tumors are attacked mainly by macrophages, whereas large highly antigenic tumors can be eradicated by cytotoxic T lymphocytes only.     

Broadening of epitope recognition during immune rejection of ErbB-2-positive tumor prevents growth of ErbB-2-negative tumor

Tumor heterogeneity is a limiting factor in Ag-specific vaccination. Ag-negative variants may arise after tumor cells bearing the immunizing Ags are destroyed. In situ priming to tumor-associated epitopes distinct from and not cross-reactive with the immunizing Ags may be crucial to the ultimate success of cancer vaccination. Immunization of BALB/c mice with DNA encoding wild-type human ErbB-2 (Her-2/neu, E2) or cytoplasmic ErbB-2 (cytE2), activated primarily CD4 or CD8 T cells, respectively, and both vaccines protected against ErbB-2-positive D2F2/E2 tumors. In > or =50% of protected mice, a second challenge of ErbB-2-negative D2F2 tumor cells was rejected. Recognition of non-ErbB-2, tumor-associated Ags was demonstrated by immune cell proliferation upon stimulation with irradiated D2F2 cells. This broadening of epitope recognition was abolished if CD4 T cells were depleted before D2F2/E2 tumor challenge, demonstrating their critical role in Ag priming. Similarly, mice that rejected D2F2/cytE2 tumor cells, which express only MHC I epitopes of ErbB-2, were not protected from a second challenge with D2F2 cells. Depletion of CD8 T cells abolished protection against D2F2, indicating the activation of D2F2-specific CTL. Therefore, long term protection may be achieved by immunization with dominant Ag(s), followed by a general enhancement of CD4 T cell activity to promote priming to multiple tumor-associated Ags.     

Adherent cell function in murine T lymphocyte antigen recognition. III. A macrophage-mediated immune response gene function in the mouse.

The I region of the MHC appears to control antigen-specific macrophage-T lymphocyte interaction. The immune response to antigens such as Gl phi 9 are under control of two distinct I subregions, I-A and I-E/I-C. We have asked in a macrophage-dependent, antigen-specific murine T cell proliferation assay whether either or both gene products need be expressed in the antigen-presenting cells. We find that both Ir-Gl phi 9 alpha and beta genes must be expressed and function in the antigen-presenting cell.     

New antigens presented on tumor cells can cause immune rejection without influencing the frequency of tumor-specific cytolytic T cells

The specific immune response against syngeneic tumors by T cells is dependent on the existence of tumor-associated transplantation antigens (TATA). In the case of the chemically induced DBA/2-derived lymphoma Eb and its highly metastatic variant ESb the immunogenicity of these antigens is not sufficient to prevent tumor growth. Therefore we tested in two systems the influence of additional antigens as possible helper determinants for the generation of tumor-specific immune responses. In the Eb tumor system additional antigens were induced by mutagenization. The frequency of cytotoxic T lymphocytes (CTL) in response to mutagenized Eb cells was higher than that in response to untreated Eb cells. Fine specificity analysis revealed there there was no increase in the CTL response against the original TATA, but an activation of additional CTL clones responding to mutagen-induced antigens. In the ESb tumor system we tested the effect of additional recognition of minor histocompatibility antigens on the frequency of TATA-specific CTL. Transplantation of ESb tumor cells into B10.D2 mice, which are H-2-identical but differ in minor antigens, results in strong tumor rejection responses. In a limiting dilution mixed-leukocyte-tumor microculture system it was found that the minor antigens are recognized at the clonal level as independent antigens. The overall frequency of anti-tumor CTL in ESb-immunized B10.D2 mice was about 1/3000. Among these, the frequency of TATA-specific CTL was 1/16,709 and thus not significantly different from that of syngeneic DBA/2 mice. Thus neither minor antigens nor mutagen-induced antigens acted in the Eb/ESb tumor system as helper determinants and did not increase the frequency of tumor-specific CTLs.     

CD8+ T cells circumvent immune privilege in the eye and mediate intraocular tumor rejection by a TNF-alpha-dependent mechanism

Although intraocular tumors reside in an immune-privileged environment, T cells can circumvent immune privilege and mediate tumor rejection without inducing damage to normal ocular tissue. In this study, we used a well-characterized tumor, Ad5E1 (adenovirus type 5 early region 1), to analyze the role of CD8+ T cells in the pristine rejection of intraocular tumors. It has been previously documented that Ad5E1 tumor rejection can occur in the absence of CD8+ T cells. However, here we find that CD8+ T cells infiltrated intraocular Ad5E1 tumors in C57BL/6 mice. Surprisingly, CD8+ T cells from tumor-rejector mice could mediate intraocular tumor rejection following adoptive transfer to SCID mice. In determining the mechanisms behind CD8+ T cell-mediated tumor rejection, we discovered that antitumor CTL activity was neither observed nor necessary for rejection of the intraocular tumors. CD8+ T cells from rejector mice did not produce IFN-gamma in response to Ad5E1 tumor Ags or use FasL to mediate intraocular tumor rejection. Also, CD8+ T cells did not use perforin or TRAIL, as CD8+ T cells from perforin knockout (KO) and TRAIL KO mice conferred protection to SCID recipient mice following adoptive transfer. We discovered that CD8+ T cells used TNF-alpha to mediate tumor rejection, because Ad5E1 tumor cells were highly sensitive to TNF-alpha-induced apoptosis and CD8+ T cells from TNF-alpha KO mice did not protect SCID mice from progressive Ad5E1 tumor growth. The results indicate that CD8+ T cells circumvent immune privilege and mediate intraocular tumor rejection by a TNF-alpha-dependent manner while leaving the eye intact and vision preserved.     

Early postnatal development of the immune system in piglets: the redistribution of T lymphocyte subsets

In this study, we have characterised postnatal changes in T lymphocyte subsets, especially γδ T lymphocytes, in blood, spleen and lymph nodes. Detection was carried out using two-colour flow cytometry and three-colour immunohistochemistry. During ontogeny, there was a significant increase in the total percentage of γδ T cells in the spleen and blood. In the lymph nodes, there were no age-dependent changes in the total percentage of γδ T cells, but the percentage of the γδTCR⁺CD8⁺ subpopulation significantly increased. The tissue distribution of γδTCR⁺CD8⁺ and γδTCR⁺CD8⁻ cells in the lymph nodes is random and not collocated with a particular area of the organ. Furthermore, postnatal development was characterised by an increasing frequency of CD8⁺CD3⁺CD4⁻γδTCR⁻, which was compensated by a decreasing proportion of CD4⁺ lymphocytes. Double positive CD4⁺CD8⁺ lymphocytes were rare during the first month of life and a significant age-dependent increase of these cells was found in all the compartments monitored.     

In situ phage screening. A method for identification of subnanogram tissue components in situ

We have established a novel method, in situ phage screening (ISPS), to identify proteins in tissue microstructures. The method is based on the selection of repertoires of phage-displayed antibody fragments with small samples of tissues microdissected using a laser. Using a human muscle frozen section with an area of 4800 microm2 as a model target, we successfully selected monoclonal antibody fragments directed against three major (myosin heavy chain, actin, and tropomyosin-alpha) and one minor (alpha-actinin 2) muscle constituent proteins. These proteins were present in the sample in amounts less than one nanogram, and the antibodies were used to visualize the proteins in situ. This shows that the use of ISPS can obtain monoclonal antibodies for histochemical and biochemical purposes against minute amounts of proteins from microstructures with no requirement for large amounts of samples or biochemical efforts.     

Toxoplasma gondii: lymphocyte function during acute infection in mice

T-cell function during acute Toxoplasma gondii infection was evaluated in murine models. Blastogenic response to the T-cell mitogen concanavalin A (Con A) was not depressed during infection with either the C37 or the C56 strain of T. gondii in either $\text{BALB}\text{c}$ or $\text{C}\text{57}\text{BL}\text{6}\text{J}$ mice that were inoculated either intravenously or intraperitoneally with varying doses of tachyzoites 7, 14, or 30 days earlier. In evaluation of lymphocytes from individual mice, utilization of a range of concentrations of Con A was found to be important for correct interpretation of results. There was variability in the magnitude of response of individual mice and in the concentration of mitogen that produced an optimal response among the inbred mice. The T-cell-dependent, primary antibody response to sheep red blood cells (SRBC) was not depressed in $\text{BALB}\text{c}$ mice infected with the C37 strain of Toxoplasma 1 and 8 days prior to inoculation with SRBC. A lower blastogenic reponse to Con A of lymphocytes from $\text{C}\text{57}\text{BL}\text{6}\text{J}$ mice compared with that of $\text{BALB}\text{c}$ mice appeared to correlate with increased susceptibility of $\text{C}\text{57}\text{BL}\text{6}\text{J}$ mice to low-challenge inocula of T. gondii.     

Target identification of biologically active small molecules via in situ methods

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Imbalance of peripheral follicular helper T lymphocyte subsets in active vitiligo

Vitiligo is an autoimmune disease characterized by the presence of several autoantibodies, some of which are directed against melanocyte components and have been shown to be associated with the progression of the disease. However, the mechanism involved in the production of autoantibodies remains unclear. Follicular helper CD4⁺ T cells (TFH) are specialized in B‐cell activation and antibody production, especially the TFH cell subsets type 2 and type 17. To date, TFH cell subsets have not been studied in human vitiligo. This study in 44 vitiligo patients and 19 healthy controls showed an increase in circulating TFH cells associated with disease clinical progression. A more precise analysis of TFH cell phenotype demonstrated that vitiligo is characterized by populations of peripheral TFH cells responsible for helping B‐cell function, such as TFH type 2 and type 17 which produce Th2‐ and TH17‐related cytokines, respectively. These findings suggest a new mechanism involving TFH cell subsets in the pathogenesis of human vitiligo and leading to the production of autoantibodies and disease.     

Visualization of polyoma virus-specific CD8+ T cells in vivo during infection and tumor rejection.

T cells are critical for clearing infection and preventing tumors induced by polyoma virus, a natural murine papovavirus. We previously identified the immunodominant epitope for polyoma virus-specific CTL in tumor-resistant H-2k mice as the Dk-restricted peptide, MT389-397, derived from the polyoma middle T oncoprotein. In this study, we developed tetrameric Dk complexes containing the MT389-397 peptide to directly visualize and enumerate MT389-397-specific CTL during polyoma virus infection. We found that Dk/MT389 tetramer+CD8+ T cells undergo a massive expansion during primary infection such that by day 7 postinfection these Ag-specific CD8+ T cells constitute approximately 20% of the total and approximately 40% of the activated CD8+ T cells in the spleen. This expansion of Dk/MT389 tetramer+CD8+ T cells parallels the emergence of MT389-397-specific ex vivo cytolytic activity and clearance of polyoma virus. Notably, Dk/MT389 tetramer+CD8+ T cells are maintained in memory at very high levels. The frequencies of Dk/MT389 tetramer+CD8+ effector and memory T cells in vivo match those of CD8+ T cells producing intracellular IFN-gamma after 6-h in vitro stimulation by MT389-397 peptide. Consistent with preferential Vbeta6 expression by MT389-397-specific CD8+CTL lines and clones, Dk/MT389 tetramer+CD8+ T cells exhibit biased expression of this Vbeta gene segment. Finally, we show that Dk/MT389 tetramer+CD8+ T cells efficiently infiltrate a polyoma tumor challenge to virus-immune mice. Taken together, these findings strongly implicate virus-induced MT389-397-specific CD8+ T cells as essential effectors in eliminating polyoma-infected and polyoma-transformed cells in vivo.     

Adaptive immune defects and delayed rejection of allogeneic tumor cells in beige mice

The effect of the beige (bg) mutation on adaptive allogeneic tumor rejection was examined by monitoring tumor cell survival in vivo using [131I]iododeoxyuridine-prelabeled cells. Accelerated elimination of allogeneic tumor cells normally begins 8 days after ip injection and is due to active immune responses. Two independent mutations to beige on two different inbred backgrounds (C57BL/6J bgJ and DBA/2JCo bg8J) were tested, and bg/bg mice showed a 1-day delay in immune elimination of allogeneic cells. This delayed rejection was not due to a defect in clearing label from dead cells, nor to an inability to effect antibody-induced killing in vivo. Both humoral and cell-mediated responses against the allogeneic tumor cells were significantly lower in bg/bg than in +/bg mice.     

Immunoregulatory function of T cells activated in the autologous mixed lymphocyte reaction

This study was undertaken to define the functional properties of T cells stimulated in the autologous mixed lymphocyte reaction (MLR) by purified B cells or macrophages. In preliminary experiments, it was found that T cells that had been cultured with autologous non-T cells inhibited pokeweed mitogen- (PWM) stimulated immunoglobulin synthesis by autologous B cells. In addition, the T cell-mediated suppression was eliminated by x-irradiation and hydrocortisone treatment, was mediated by a mechanism that occurred early in the PWM-stimulated cultures, and did not involve killing of mature immunoglobulin-secreting cells. T cells were then cultured with either autologous B cells or macrophages in order to determine whether such autoreactive T cells had a similar capacity to regulate PWM-induced immunoglobulin synthesis. Although T cell populations stimulated either by B cells or by macrophages suppressed proliferative responses and immunoglobulin synthesis, both these populations of autoreactive T cells provided help for immunoglobulin synthesis that was not significantly different from that provided by fresh T cells. These results suggest that the predominant functional consequence of activation of T cells in the autologous MLR is the generation of suppressor T cells capable of inhibiting immunoglobulin synthesis. Thus, the autologous MLR may represent a negative feedback mechanism for the regulation of the immune response.