Presence Of anti-CD3 Antibody Research Articles

Abstract 6373: P-selectin glycoprotein ligand-1 modulates the functions of human T cells and macrophages in vitro

Abstract P-selectin glycoprotein ligand-1 (PSGL-1) is a type I transmembrane protein expressed on the surface of most hematopoietic cells. PSGL-1 can engage multiple ligands (e.g., selectins, VISTA, Siglec-5, versican, CCL19, and CCL21). Apart from being a key adhesion molecule involved in immune cell trafficking, PSGL-1 has been shown to function as a negative immune checkpoint receptor in both T cells and macrophages. PSGL-1 is highly expressed in tumor-infiltrating T cells (TILs) and in tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). PSGL-1 signaling in TILs induces development of T-cell exhaustion and, in TAMs, it promotes immunosuppressive activity of macrophages. In the TME, PSGL-1 is also highly expressed on the surface of myeloid-derived suppressor cells, regulatory T cells, and some types of cancer cells. We hypothesized that PSGL-1 may also act as a ligand to modulate the effector functions of human T cells and macrophages in the TME. In the present study, flow cytometry analysis showed that monocytes and granulocytes had a higher PSGL-1 expression than CD4+ and CD8+ T cells isolated from normal human blood cells. To mimic the cell-expressing PSGL-1, a recombinant human PSGL-1-Fc protein (rhPSGL-1-Fc, R&D Systems) was coated onto 96-well plates overnight. Human peripheral blood mononuclear cells (PBMCs), purified human CD4+ T cells, or CD8+ T cells were cultured in the PSGL-1-coated plates for 3 days in the presence of anti-CD3 antibody. In some experiments, a commercial tool PSGL-1 monoclonal antibody (mAb) with the ability to block PSGL-1/P-selectin interaction was added into the culture system. Cytokine secretion in the culture supernatants was measured using Meso Scale Discovery assays. Similar experiments were also conducted with commercially supplied human M1 and M2 macrophages. The results demonstrated that plate-bound rhPSGL-1-Fc dose-dependently inhibited IFN-γ, IL-2, and TNF-α cytokine production in human PBMCs, CD4+ T cells, and in CD8+ T cells following T-cell receptor stimulation. The tool PSGL-1 mAb was able to partially rescue the suppression of cytokine production. Plate-bound rhPSGL-1-Fc also inhibited TNF-α secretion from human M1 macrophage, and the PSGL-1 mAb significantly enhanced TNF-α production in both human M1 and M2 macrophages treated with plate-bound PSGL-1-Fc. Together, these data indicate that PSGL-1 expressed on immune cells in TME may act as both an inhibitory ligand and receptor to impact on T-cell and macrophage function. Acknowledgements: Thanks to Patrick Mayes and Ricardo Macarron for scientific input and thanks to Qian Wang Yao and Lynn Leffet for technical assistance. Trial Registration: N/A Ethics Approval: N/A Citation Format: Jun Guan, Sundee Dees, Tony Chadderton, Alejandro Amador Arjona. P-selectin glycoprotein ligand-1 modulates the functions of human T cells and macrophages in vitro. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6373.

GAMMA-DELTA T CELLS MEDIATE ALPHA-BETA T CELL ACTIVATION IN ANGIOTENSIN II-INDUCED HYPERTENSION

Objective: Both innate and adaptive immune cells have been shown to play a role in hypertension and vascular injury. Recently, we demonstrated that a small subset of “innate-like” T lymphocytes, expressing the γδ T cell receptor (TCR) rather than the αβ TCR, plays a key role in hypertension and vascular injury. We demonstrated an increased number and activation (CD69+) of αβ and γδ T cells during the development of hypertension caused by angiotensin (Ang) II infusion, and that deficiency in γδ T cells prevented Ang II-induced hypertension, resistance artery endothelial dysfunction and spleen T-cell activation in mice. We hypothesized that γδ T cells mediate activation of other T cells in hypertension. Design and method: Fourteen to 15-week-old aged C57BL/6J male mice were infused or not with Ang II (490 ng/kg/min, SC) for 7 and 14 days (n = 5–7). γδ and αβ T cells were isolated from peripheral lymph nodes and spleen and αβ T cells were cultured alone or with γδ T cells (5:1) in presence of anti-CD3 antibodies plus or minus Ang II. T cell phenotype was evaluated by flow cytometry. Results: Close correlations were demonstrated between the number (#) of activated CD69 + γδ T cells and CD4 + CD69 + T cells (r2 = 0.74, P < 0.01) and CD8 + CD69 + T cells (r2 = 0.64, P < 0.01) after 7-day Ang II infusion. In vitro, Ang II increased the fraction of CD69 + αβ T cells when αβ T cells were co-cultured with γδ T cells isolated from control mice (% of αβ T cells: 19.9 ± 2.9 vs. 16.4 ± 2.6, P < 0.001) but not when cultured alone. The fractions of CD69 + (% of CD69 + αβ T cells: 35.7 ± 2.7 vs. 18.9 ± 1.5, P < 0.001) and CCR6 + αβ T cells (% of CCR6 + αβ T cells: 27.6 ± 2.4 vs. 19.2 ± 3.5, P < 0.05) were increased when αβ T cells were co-cultured with γδ T cells isolated from Ang II-infused compared to control mice. Conclusions: These results suggest that γδ T cells mediate activation of αβ T cells in Ang II-induced hypertension. Targeting γδ T cells may contribute to reduce the low-grade inflammation found in hypertension.

Early Reconstitution of CD6+ T Cells after Hematopoietic Cell Transplantation Identifies a Suitable Target for Acute Graft Versus Host Disease Treatment Using Anti-CD6 Monoclonal Antibody Itolizumab

Early Reconstitution of CD6+ T Cells after Hematopoietic Cell Transplantation Identifies a Suitable Target for Acute Graft Versus Host Disease Treatment Using Anti-CD6 Monoclonal Antibody Itolizumab

Abstract 023: γδ T Cells Mediate αβ T Cell Activation in Angiotensin II-Induced Hypertension

Objectives: Both innate and adaptive immune cells have been shown to play a role in hypertension and vascular injury. Recently, we demonstrated that a small subset of “innate-like” T lymphocytes, expressing the γδ T cell receptor (TCR) rather than the αβ TCR, plays a key role in hypertension and vascular injury. We demonstrated an increased number and activation (CD69 + ) of αβ and γδ T cells during the development of hypertension caused by angiotensin (Ang) II infusion, and that deficiency in γδ T cells prevented Ang II-induced hypertension, resistance artery endothelial dysfunction and spleen T-cell activation in mice. We hypothesized that γδ T cells mediate activation of other T cells in hypertension. Methods: C57BL/6 male mice were infused with Ang II (490 ng/kg/min, SC) for 7 and 14 days (n=5-7). All mice were 14-15 week-old at the end of the study. Spleen T cell profile was determined by flow cytometry. γδ and αβ T cells were isolated using magnetic beads from peripheral lymph nodes and spleen from C57BL/6 male mice treated or not with Ang II for 14 days. αβ T cells were cultured alone or with γδ T cells (5:1) in presence of anti-CD3 antibodies plus or minus Ang II, and αβ T cell phenotype was evaluated by flow cytometry. Results: Close correlations were demonstrated between the number (#) of activated CD69 + γδ T cells and CD4 + CD69 + T cells (r 2 =0.74, P <0.01) and CD8 + CD69 + T cells (r 2 =0.64, P <0.01) after 7-day Ang II infusion. Correlations were also shown between the # of CD27 + CD69 + γδ T cells and CD4 + CD69 + T cells (r 2 =0.76, P <0.001) and CD8 + CD69 + T cells (r 2 =0.65, P <0.01) after 7-day Ang II infusion. In vitro , Ang II increased the fraction of CD69 + αβ T cells when αβ T cells were co-cultured with γδ T cells isolated from control mice (% of αβ T cells: 19.9±2.9 vs. 16.4±2.6, P <0.001) but not when cultured alone. The fractions of CD69 + (% of CD69 + αβ T cells: 35.7±2.7 vs. 18.9±1.5, P <0.001) and CCR6 + αβ T cells (% of CCR6 + αβ T cells: 27.6±2.4 vs. 19.2±3.5, P <0.05) were increased when αβ T cells were co-cultured with γδ T cells isolated from Ang II-infused compared to control mice. Conclusions: These results suggest that γδ T cells mediate activation of αβ T cells in Ang II-induced hypertension. Targeting γδ T cells may contribute to reduce the low-grade inflammation found in hypertension.

Recurrent inflammatory disease caused by a heterozygous mutation in CD48

Recurrent inflammatory disease caused by a heterozygous mutation in CD48

Abstract 2397: Pre-clinical evaluation of PE0116: A potent cross-linking depednet 4-1BB agonist antibody

Abstract 4-1BB is a member of the tumor necrosis factor superfamily and plays a critical role in activation of cytotoxic T cells to promote anti-tumor immunity. As a co-stimulatory molecule, it is ideally suited for activation via agonist therapeutic antibodies in order to reactivate the immune system and promote cytolytic activity through CD8+ T cells. Clinical trials of two agonist antibodies (Urelumab and Utomilumab) are ongoing. Urelumab has shown clinical efficacy but been hampered by inflammatory liver toxicity at doses above 1 mg/kg, suggesting potential safety concerns at higher doses. Utomilumab has a superior safety profile, but is a less potent 4-1BB agonist. Hence, it may be necessary to achieve potent immune activation while avoiding limiting immune-related adverse events. PE0116 is a fully human monoclonal antibody generated from immunization of Harbour H2L2 human transgenic mice with recombinant human 4-1BB protein. It blocks the binding of 4-1BB to its ligand 4-1BBL and its activity in T cell activation is crosslinking dependent. After crosslinking, PE0116 strongly increases IFNg secretion from T cells in the presence of anti-CD3 antibody and its activity is superior to Utomilumab analog. More importantly, PE0116 has demonstrated robust anti-tumor activity and better potency than both benchmark antibodies in MC38 tumor model with human 4-1BB KI mice. PE0116 has also demonstrated good safety profile in a non-GLP safety study in cyno monkey. Currently the antibody is under late stage IND-enabling study. Its safety and efficacy will be further tested in patients. Citation Format: Ningning Song, Zhengwu Li, Shan He, Teddy Yang, Qing Duan. Pre-clinical evaluation of PE0116: A potent cross-linking depednet 4-1BB agonist antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2397.

A Critical Role of Host-Derived Semaphorin-4A for Regulating T Cell Immune Responses in Acute Graft Versus Host Disease

Semaphorins, originally identified as repulsive axon-guidance factors that participate in neuronal development. Several semaphorins are involved in various phases of immune responses, as has been shown recently. Semaphorin4A (Sema4A), a class IV transmembrane semaphorin, is preferentially expressed in dendritic cells (DCs) and Th1 cells. Previous studies suggested that Sema4A is involved in Ag-specific T cell priming, and in Th1 cell and Th17 cell differentiation. Additionally, Sema4A is required for the stability and function of Tregs. However, the role of Sema4A in alloimmune responses remains to be elucidated. In this study, we examined the contribution of Sema4A to T cell immune responses in the context of allogeneic hematopoietic stem cell transplantation (allo-HCT).To test the role of Sema4A in T cell proliferation, we performed in vitro T cell proliferation assay using dendritic cells harvested from wild-type or Sema4A-KO mice. Conventional CD4+T cells cocultured with DCs harvested from wild-type and Sema4A-KO mice proliferated equally in the presence of anti-CD3 antibody. In contrast, anti-CD3-induced proliferation of Tregs cocultured with DCs harvested from Sema4A-KO mice was attenuated compared to Tregs cocultured with DCs harvested from wild-type mice, suggesting that Sema4A is required for maximum proliferation of Tregs.We next investigated the effects of Sema4A deficiency in acute GVHD. We employed a murine acute GVHD model (B6→BALB/c) and monitored every day for survival. Body weight was assessed 2-3 times per week. First, to investigate the role of host Sema4A, lethally irradiated wild-type and Sema4A-KO mice were injected with T cell-depleted bone marrow cells and T cells isolated from wild-type B6. Sema4A-KO host mice developed significantly higher mortality compared to wild-type host mice (p<0.001) (Figure). On day 7 after allo-HCT, the proportion of Tregs in the spleen were significantly decreased in Sema4A-KO host mice compared to wild-type host mice (p<0.05). Both donor-derived preexisting natural Tregs and inducible Tregs in Sema4A-KO host mice were significantly decreased compared to wild-type host mice, although relatively less in inducible Tregs. We investigated the production of proinflammatory cytokines (IFN-g, IL-17, IL-4 and IL-13) of T cells in the spleen by intracellular cytokine staining. The proportion of IL-17+CD4+ T cells in Sema4A-KO host was slightly but not significantly decreased compared to wild-type host, whereas similar proportion of IFN-g+CD4+T cells was observed in Sema4A-KO and wild-type host mice. In contrast, the proportion of IL-4+CD4+ T cells in Sema4A-KO host was significantly increased in Sema4A-KO host mice compared to wild-type host mice, suggesting Sema4A deficiency skewed cytokine polarization of T cells after allo-HCT.Subsequently, we used Sema4A-KO and wild-type B6 mice donor to investigate the role of donor-derived Sema4A. Host mice transplanted from Sema4A-KO donor developed comparable mortality with host mice transplanted with wild-type donor, suggesting that donor-derived Sema4A does not play an important role in controlling graft versus host reactions in this model.Together, these results suggest that Sema4A contributes to Treg maintenance and the regulation of T cell responses after allo-HCT, which may have clinical implications for the novel approach in the treatment and protection of GVHD. [Display omitted] DisclosuresIto:Bristol-Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria; Novartis Pharma: Honoraria; Pfizer: Honoraria; Mundipharma: Honoraria; Celgene: Honoraria.

Abstract P283: Gamma Delta T Cells Drive CD4 + and CD8 + T Cell Activation in Hypertension

Objective: Both innate (monocyte/macrophages) and adaptive immune cells (T lymphocytes) have been shown to play a role in hypertension and vascular injury. Recently, we demonstrated that a small subset of “innate-like” T lymphocytes, expressing the γδ T cell receptor (TCR) rather than the αβ TCR, plays a key role in hypertension and vascular injury. We demonstrated an increased number and activation (CD69 + ) of γδ T cells during the development of hypertension caused by angiotensin (Ang) II infusion, and that deficiency in γδ T cells prevented Ang II-induced hypertension, resistance artery endothelial dysfunction and spleen T-cell activation in mice. We hypothesized that γδ T cells mediate activation of other T cells in hypertension. Method: C57BL/6 male mice were infused with Ang II (490 ng/kg/min, SC) for 7 and 14 days (n=5-7). All mice were 14-15 week-old at the end of the study. Spleen T cell profile was determined by flow cytometry. γδ and αβ T cells were isolated using magnetic beads from peripheral lymph nodes and spleen of 8 to 9-week-old C57BL/6 male mice. αβ T cells were cultured alone or with γδ T cells (5:1) in presence of anti-CD3 antibodies plus or minus Ang II, and αβ T cell activation was evaluated by flow cytometry. Results: Close correlations were demonstrated between the number (#) of activated CD69 + γδ T cells and CD4 + CD69 + T cells (r 2 =0.74, P &lt;0.01) and CD8 + CD69 + T cells (r 2 =0.64, P &lt;0.01) after 7-day Ang II. These correlations decreased after 14-day Ang II. Correlations were also shown between the # of CD27 + CD69 + γδ T cells and CD4 + CD69 + T cells (r 2 =0.76, P &lt;0.001) and CD8 + CD69 + T cells (r 2 =0.65, P &lt;0.01) after 7-day Ang II. In vitro , Ang II increased the fraction of CD69 + αβ T cells when αβ T cells were co-cultured with γδ T cells (% of αβ T cells: 19.9±2.9 vs. 16.4±2.6, P &lt;0.001) but not when cultured alone (% of αβ T cells: 13.7±0.7 vs. 12.7±0.6). A correlation between the fraction of CD69 + γδ T cells and CD69 + αβ T cells was also observed (r 2 =0.69, P &lt;0.001). Conclusion: These results suggest that γδ T cells mediate activation of αβ T cells in Ang II-induced hypertension. Targeting γδ T cells may contribute to reduce the low-grade inflammation found in hypertension.

Expression of ergotope-associated markers of T lymphocytes in atopic dermatitis after in vitro polyclonal activation

The antiergotypic response leads to the formation of effector T cells able to eliminate activated lymphocytes independently of their antigenic specificity, since the targets of these cells are molecules produced during cell activation (ergotopes). In this paper, we describe the level of expression of the ergotope-associated markers CD25, HSP60, and HLA-DR by the T lymphocytes isolated from the blood of atopic dermatitis patients immediately after isolation and after cultivation. After 10-day cultivation in the presence of anti-CD3 antibodies and IL-2, the expression levels of early and late activation markers in T cells have changed: the shares of CD25-positive CD4+ and CD8+ lymphocytes increase to 68 and 47%, respectively, and the share of HLA-DR-positive cells increases to 26 and 33%. The density of HLA-DR molecules on the surface of activated T cells increases more than fivefold. Almost all T cells before and after cultivation express 60 kDa heatshock protein (HSP60); however, the CD4+ cells activated in vitro contain more HSP60 molecules than do the in vitro-activated CD8+ cells and the CD4+ cells of peripheral blood. Thus, the T cells of atopic-dermatitis patients have the status of activated cells because they express sufficient amounts of early and late activation markers; presumably, they can enhance the induction of antiergotypic response when administered to patients. Taking into account that antiergotypic regulation acts on activated T cells independently of their antigenic specificity, immunotherapy utilizing autologous activated T lymphocytes can be of interest as a method for targeted action on pathogenetic components of atopic dermatitis.

Preclinical Efficacy and Immunological Safety of FR104, An Antagonist Anti-CD28 Monovalent Fabʼantibody, in Humanized Mice Models

Antagonist anti-CD28 antibodies prevent T-cell costimulation and functionally differentiate from CTLA4Ig since they cannot block CTLA-4, PDL-1 and CD80/86-mediated coinhibitory signals. They demonstrated efficacy in suppressing effector T cells while enhancing regulatory T cells function and immune tolerance after organ transplantation. So far, however, blocking anti-CD28 antibodies devoid of any agonist or superagonist activity and showing clinically acceptable pharmacokinetic profile have not been developed. FR104 is a novel monovalent humanized Fab' anti-CD28 antibody fragment that was pegylated to improve elimination half-life. We have investigated its mechanism of action, immunological safety and preclinical efficacy in humanized mouse models. FR104 dose-dependently prevented human T cell proliferation and IL-2 secretion in vitro. Following the guidelines edicted after the ‘Tegenero affair’ to exclude agonist activities of T-cell directed antibodies, we showed that FR104 failed to induce human T cell proliferation and cytokines secretion, even in the presence of anti-CD3 antibodies or when cross-linked with secondary antibodies, whereas superagonist and divalent anti-CD28 antibodies showed such activities. Furthermore, in humanized NOD/SCID mice adoptively transferred with human PBMC, superagonist and divalent anti-CD28 Abs elicited rapid cytokines secretion and human T-cell activation, whereas FR104 stayed antagonist. These humanized mice eventually developed a florid graft-versus-host disease due to the proliferation of xenogeneic human T cells, that was completely prevented by short-term administration of FR104. This effect was dependent on the availability of CTLA-4 since co-administration of an anti-CTLA-4 antibody abrogated the therapeutic activity of FR104. Interestingly, administration of high doses of Belatacept was ineffective to prevent graft-versus-host disease, whereas a less intensive regimen of administration was partially effective. In conclusion, we demonstrated in vitro and in vivo on human T cells that the anti-CD28 monovalent antibody FR104 is devoid of agonist activity and thus compatible with a clinical development that might lead to higher therapeutic indexes, by sparing CTLA-4, as compared to CD80/CD86 antagonists in autoimmune diseases and transplantation.

Preclinical Efficacy and Immunological Safety of FR104, an Antagonist Anti-CD28 Monovalent Fab′ Antibody

Antagonist anti-CD28 antibodies prevent T cell costimulation and differentiate from CTLA4Ig since they cannot block CTLA-4 and PDL-1 coinhibitory signals. They demonstrated efficacy in suppressing effector T cells while enhancing regulatory T cells function and immune tolerance. However, anti-CD28 antibodies devoid of immunotoxicity and with a good pharmacokinetic profile have not yet been developed. Here, we describe FR104, a novel humanized pegylated anti-CD28 Fab' antibody fragment presenting a long elimination half-life in monkeys. In vitro, FR104 failed to induce human T cell proliferation and cytokines secretion, even in the presence of anti-CD3 antibodies or when cross-linked with secondary antibodies. Furthermore, in humanized NOD/SCID mice adoptively transferred with human PBMC, whereas superagonist and divalent antibodies elicited rapid cytokines secretion and human T cell activation, FR104 did not. These humanized mice developed a florid graft-versus-host disease, which was prevented by administration of FR104 in a CTLA4-dependent manner. Interestingly, administration of high doses of CTLA4-Ig was ineffective to prevent GVHD, whereas administration of low doses was partially effective. In conclusion, we demonstrated that FR104 is devoid of agonist activity on human T cells and thus compatible with a clinical development that might lead to higher therapeutic indexes, by sparing CTLA-4, as compared to CD80/CD86 antagonists.

Adaptive immune responses during Shigella dysenteriae type 1 infection: an in vitro stimulation with 57 kDa major antigenic OMP in the presence of anti-CD3 antibody

An effort was made to understand the role of the 57 kDa major antigenic fraction of Shigella outer membrane protein (OMP) in the presence of T-cell antigen receptor in activation of adaptive immune responses of the cell mediated immune (CMI) restored patients. The expression of HLA-DR/CD4 out of CD3(+) T-cells was significantly dominant over the HLA-DR/CD8 and comparable to unstimulated cells of infected or healthy controls. CD4(+) T-cell activation together with HLA-DR is associated with the expression of CD25(+) (IL2Ralpha) for IL-2 growth factors with decreased IL-4 levels, required for maintaining the homeostasis of CD4(+) T cell. Furthermore, the positive expression of the CD45 antigen is possibly required for acquiring the memory for CD4(+) cells signals and facilitates the interaction with CD54 antigen. As a result, antigen-specific secondary signal is generated for B-cell activation to produce IgG2a and IgG2b. This suggests that antibody mediated-adaptive immune responses are generated due to anti-CD3 induced helper T-cell activity. The above mentioned findings reflect that the antigen alone might not exacerbate the selective T-cell responses. But these antigens in the presence of anti-CD3 antibody might help to elicit adaptive immune response via T-cell receptor (TCR) activation.

Immunologic Properties Differ in Preterm Infants Fed Olive Oil vs Soy‐Based Lipid Emulsions During Parenteral Nutrition

In the first period of life, premature infants need parenteral nutrition. Lipid emulsions (LEs), which are a part of parenteral nutrition, are known as potent immunological modulators and may therefore influence the immune status of parenterally fed infants. The aim of the study was to compare tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-10 production in the peripheral blood mononuclear cells (PBMCs) of premature infants parenterally fed with 2 LEs: olive oil (OO) and soybean oil (SO). Premature infants born at <32 weeks' gestation and with a birth weight <1500 g were randomized in a double-blind method within the first 48 hours of life to receive 1 of 2 LEs: OO based or SO based. At baseline and after 14 days, blood samples were collected, and PBMCs were isolated and then cultured for 48 hours in medium only and in the presence of anti-CD3 antibodies. Of 44 recruited infants, 38 completed the study, 18 in the OO group and 20 in the SO group. The cytokine synthesis profile before the LE introduction was the same in both groups (nonstimulated and anti-CD3-induced PBMC). In the succeeding 14 days of parenteral nutrition, TNF-alpha, IL-6, and IL-10 levels in nonstimulated PBMCs remained unchanged in both groups. In contrast, IL-6 production was significantly higher in the SO group. SO-based LE may promote an excess of IL-6 production, especially in the T cell-dependent way of PBMC activation (via anti-CD3). OO emulsion seems to be immunologically more neutral than SO emulsion.

Inhibition of human ovarian tumor growth by cytokine-induced killer cells

Despite the recent improvement in the treatment of ovarian cancer, this disease is still leading cause of cancer death in women. In this study, the anti-tumor activity of cytokine-induced killer (CIK) cells against human ovarian cancer was evaluated in vitro and in vivo. Although CD3+CD56+ cells were rare in fresh human peripheral blood mononuclear cells, they could expand more than 1,000-fold on day 14 in the presence of anti-CD3 antibody plus IL-2. At an effector-target cell ratio of 30:1, CIK cells destroyed 45% of SK-OV-3 human ovarian cancer cells, which was determined by the 51Cr-release assay. In addition, CIK cells at a dose of 23 million cells per mouse inhibited 73% of SK-OV-3 tumor growth in nude mouse xenograft assay. This study suggests that CIK cells may be used as an adoptive immunotherapy for patients with ovarian cancer.

Antitumor activity of cytokine-induced killer cells against human lung cancer

Lung cancer is the leading cause of cancer-related death among men and women in the world. Despite the aggressive treatment with surgery, radiation and chemotherapy, the long term survival for lung cancer patients remains low. In this study, the anti-tumor activity of cytokine-induced killer (CIK) cells against human lung cancer was evaluated in vitro and in vivo. Although CD3+CD56+ CIK cells were rare in fresh human peripheral blood mononuclear cells, they could expand more than 1000-fold on day 14 in the presence of anti-CD3 antibody plus IL-2. At an effector-target cell ratio of 30:1, CIK cells destroyed 98% of NCI-H460 human lung cancer cells, which was determined by the 51Cr-release assay. In addition, CIK cells at doses of 3 and 30 million cells per mouse inhibited 57% and 77% of NCI-H460 tumor growth in nude mouse xenograft assay, respectively. This study suggests that CD3+CD56+ CIK cells may be used as an adoptive immunotherapy for patients with lung cancer.

Anti-tumor activity of ex vivo expanded cytokine-induced killer cells against human hepatocellular carcinoma

Cytokine-induced killer (CIK) cells are ex vivo expanded T cells with natural killer cell phenotypes and functions. In this study, the anti-tumor activity of CIK cells against hepatocellular carcinoma was evaluated in vitro and in vivo. In the presence of anti-CD3 antibody and IL-2 for 14 days, human peripheral blood mononuclear cell population changed to heterogeneous CIK cell population, which comprised 96% CD3+, 3% CD3¡©CD56+, 32% CD3+CD56+, 11% CD4+, 75% CD8+, and 30% CD8+CD56+. CIK cells produced significant amounts of IFN-γ and TNF-α; however, produced only slight amounts of IL-2, IL-4, and IL-5. At an effector–target cell ratio of 30:1, CIK cells destroyed 33% of SNU-354 human hepatocellular carcinoma cells, which was determined by the 51Cr-release assay. In addition, a dose of 1×106 CIK cells per mouse inhibited 60% of SNU-354 tumor growth in irradiated nude mice. This study suggests that CIK cells may be used as an adoptive immunotherapy for patients with hepatocellular carcinoma.

Interleukin-18 restores immune suppression in patients with nonseptic surgery, but not with sepsis

Background We investigated cellular immune responses, in particular interferon γ (IFN-γ) production, by peripheral blood mononuclear cells (PBMCs) in patients with septic and nonseptic surgical stress, focusing on interleukin (IL)-18 and its receptor (IL-18R). Methods Thirty-two patients with alimentary tract carcinoma who underwent elective surgery (OP) and 26 septic patients (SP) with peritonitis were enrolled in this study. Blood was collected on the first postoperative day (POD1), POD5, POD10, and POD15 in the OP group and on the emergency admission in the SP group. Ten healthy volunteers served as controls. PBMCs were cultured in the presence of anti-CD3 antibody or IL-2 and IL-12, with or without additional IL-18 stimulation, to measure IFN-γ production. IL-18R expression on CD56 +NK (natural killer) cells was evaluated by flow cytometry. Results IL-2– and IL-12–induced IFN-γ production by PBMCs was suppressed significantly in both the OP (POD5) and SP groups compared with that in healthy controls. Interestingly, additional IL-18 stimulation up-regulated IFN-γ production by PBMCs in the OP group as well as the control group, but not in the SP group. IL-18R expression on CD56 +NK cells was maintained consistently in the OP group as well as the control group, but decreased in the SP group. Conclusions IFN-γ production induced by cytokines (IL-2 and IL-12) was suppressed in PBMCs from both patients with sepsis and those who had undergone elective surgery. However, IL-18R expression on CD56 +NK cells was different between patients with sepsis and nonseptic surgical stress. Our results suggest that exogenous IL-18 administration may be effective in preventing immune suppression in patients with nonseptic elective surgery.

Interferon-gamma-inducing factor elicits antitumor immunity in association with interferon-gamma production.

Interferon-gamma-inducing factor (IGIF) is a novel cytokine that stimulates T-cell proliferation, augments natural killer (NK) cell lytic activity, and induces interferon-gamma (IFN-gamma) production in established type 1 T-helper (Th1) cells in the presence of anti-CD3 antibody. The in vitro induction of IFN-gamma by recombinant murine IGIF in these cells was more potent than that induced by murine interleukin-12 (IL-12) and occurred apparently independent of murine IL-12. Here we report that subcutaneous injection into mice of tumor cells transfected with murine IGIF complementary DNA (cDNA) resulted in > or = 10-fold increase of mitogen-stimulated IFN-gamma production in cultured splenocytes. In addition, IGIF-transfected Renca and K1735 tumor cells can be rejected in vivo. The IGIF antitumor effect was abrogated in mice that were sublethally irradiated or depleted of both CD4+ and CD8+ T cells but not in mice depleted of either subpopulation alone. The antitumor effect mediated by IGIF appears to be dependent on IFN-gamma production, because in vivo neutralization of IFN-gamma was accompanied by growth of IGIF-transfected tumors in 100% of the animals. Taken together, our results show that murine IGIF can elicit T-cell-dependent antitumor immunity associated with IFN-gamma induction.

Infection with Trypanosoma cruzi selectively upregulates B7-2 molecules on macrophages and enhances their costimulatory activity.

T-cell-mediated immune responses are essential for protection against infection with the protozoan Trypanosoma cruzi. In this study, we investigated the influence of infection of murine macrophages with T. cruzi on costimulatory signals for T lymphocytes provided by these cells. We demonstrate that bone marrow-derived macrophages (BMMph) selectively and strongly upregulate expression of B7-2 molecules after infection, while the expression of other costimulatory molecules such as B7-1, intercellular adhesion molecule 1, lymphocyte function-associated antigen 3, and heat-stable antigen is not significantly affected. Infection by live trypanosomes was required. As a consequence of the strong B7-2 upregulation, the infected macrophages are able to induce proliferation of splenic CD4+ T cells in the presence of anti-CD3 antibodies with much higher efficiency than uninfected macrophages. Costimulation could be inhibited by an antibody to B7-2. Furthermore, costimulatory activity for established T-cell clones of Th1 and Th2 phenotype was also strongly enhanced in BMMph by infection with T. cruzi. Th1 cells stimulated either via anti-CD3 antibodies or via specific antigen proliferated with higher efficiency in the presence of infected macrophages than in the presence of uninfected cells. BMMph stimulated with gamma interferon (IFN-gamma), expressing elevated levels of B7-2 molecules, are also able to enhance Th1 cell proliferation, which was highest, using macrophages which were infected and in parallel were stimulated with IFN-gamma. Noteworthy, for cloned Th2 cells, the mechanism of costimulation differed, because costimulation of Th2 cells was not dependent on B7-2 upregulation but was due to secretion of interleukin-1alpha. These findings demonstrate that infection of macrophages with T. cruzi transforms the macrophage into a potent costimulatory cell based on the induction of two different costimulatory activities.

Association of Serine Protease with the Rise of Intracellular Calcium in Cytotoxic T Lymphocytes

The precise role of the granular enzyme A (granzyme A), a serine protease, in the lytic process of cytotoxic T lymphocytes (CTL) is not clear. We have recently constructed a CTL line transfected with the antisense gene of granzyme A (a-GrA). These a-GrA CTL had lower GrA activity as well as decreased lytic activities, as measured by51Cr and by DNA degradation assays. Furthermore, at low effector:target ratio (1:8) in prolonged lytic assays, they could not lyse targets as rapidly as the control CTL. When we examined their ability to exocytose BLT (CBZ-L-lys-thiobenzyl)-esterase in the presence of anti-CD3 antibody, the a-GrA CTL exocytosed poorly compared to the parental CTL or control transfectant with a CAT gene. Most strikingly, a-GrA cells could not release intracellular stores of Ca2+in response to anti-CD3 induction, although the Ca2+flux was normal when they were stimulated with ionomycin. When the parental CTL was treated with a specific benzyllactam inhibitor of BLT-esterase orN-tosyl-L-phenylalanylchloromethyl ketone, the Ca2+flux induced by anti-CD3 was also suppressed. We propose that granzyme A is involved in the signal transduction pathway that causes the rise of the intracellular calcium.