Effector Cells For Adoptive Immunotherapy Research Articles

Optimizing the procedure for manufacturing clinical-grade genetically manipulated natural killer cells for adoptive immunotherapy

Background aimsEx vivo−expanded natural killer (NK) cells hold significant potential as antitumor effector cells for adoptive immunotherapy. However, producing clinical-grade, genetically modified NK cells in sufficient quantities presents a considerable challenge. MethodsWe tested RPMI 1640, KBM581, SCGM, NK MACS, X-VIVO 15 and AIM-V, each supplemented with fetal bovine serum, human AB serum, human platelet lysate or Immune Cell Serum Replacement (SR) combined with feeder cells, to produce cytotoxic NK cells. Subsequent analyses were conducted to assess cell viability, expansion folds, cytotoxicity, immunophenotype and transcriptome profile of NK cells under certain conditions. Furthermore, transfer plasmids varying in transgene size, promoter elements, backbones and packaging plasmids with different envelopes were used to transduce NK cells, and differences in transduction efficiency were compared. Nucleofection was performed every 2 days from day 0 to day 12 to determine the optimal time window for gene editing. ResultsNK cells cultured in KBM581 medium supplemented with serum replacement exhibited the best expansion, achieving greater than 5000-fold increase within 2 weeks and exceeding 25 000-fold expansion within 3 weeks. In addition, NK cells cultured in KBM581 medium with human AB serum demonstrated the greatest cytolytic activities and exhibited greater expression of NKp30, 2B4, PRF1, granzyme B and IL2RG. Baboon envelope pseudotyped lentivirus outperformed baboon envelope-vesicular stomatitis virus type G hybrid envelope lentivirus, achieving robust NK-cell transduction. In addition, efficient gene knockout efficiency was achieved in NK cells on day 4 to day 6 post feeder cell activation using the LONZA DN-100 program, which can strike a balance between editing efficiency and cell expansion. ConclusionsThis research presents a Good Manufacturing Practice−compliant protocol using a feeder cell expansion system for the large-scale production of highly cytotoxic NK cells. The protocol facilitates genetic modification of these cells, positioning them as promising candidates for universal therapeutic applications in immunotherapy.

Chimeric antigen receptor natural killer cells: a promising antitumor immunotherapy.

Chimeric antigen receptor (CAR) T cells have been successfully used in adoptive cell therapy for malignancies. However, some obstacles, including side effects such as graft-versus-host disease and cytokine release syndrome, therapy resistance, limited sources, as well as high cost, limited the application of CAR T cells. Recently, CAR natural killer (NK) cells have been pursued as the effector cells for adoptive immunotherapy for their attractive merits of strong intrinsic antitumor activity and relatively mild side effects. Additionally, CAR NK cells can be available from various sources and do not require strict human leukocyte antigen matching, which suggests them as promising "off-the-shelf" products for clinical application. Although the use of CAR NK cells is restrained by the limited proliferation and impaired efficiency within the immunosuppressive tumor microenvironment, further investigation in optimizing CAR structure and combination therapies will overcome these challenges. This review will summarize the advancement of CAR NK cells, CAR NK cell manufacture, the clinical outcomes of CAR NK therapy, the challenges in the field, and prospective solutions. Besides, we will discuss the emerging application of other immune cells for CAR engineering. Collectively, this comprehensive review will provide a valuable and informative summary of current progress and evaluate challenges and future opportunities of CAR NK cells in tumor treatment.

4-1bb Enrichment Enhances Adoptive T Cell Therapy for Hematological Malignancies Using a Novel Approach for Ex Vivo Immune Cell Priming with DC/Tumor Fusion Vaccine

Introduction: While CAR T cells have transformed outcomes for patients with hematologic malignancies, application to patients with acute myeloid leukemia (AML) is limited by lack of surface antigens that differentiate between malignant and normal hematopoietic precursors. We have developed a personalized vaccine created by fusion of patient-derived tumor with autologous dendritic cells (DCs). The vaccine presents an array of tumor antigens stimulating polyclonal immunity. In a phase II clinical trial of patients with AML in first remission, vaccination with DC/AML fusions resulted in expansion of leukemia specific T cells associated with durable disease remission. The ex vivo generation of vaccine educated T cells provides a powerful substrate of effector cells for adoptive immunotherapy that are selective and capture tumor heterogeneity. This novel adoptive cell therapy enhances T-cell potency with potential to mediate disease regression. Enrichment of vaccine-educated T cells for activated antigen-specific effector cells via agonistic 4-1bb antibody-based selection further enhances for activation and memory cell phenotype; superior T cell efficacy was observed in AML and MM models. Methods: DC/tumor fusion vaccines were generated from C57BL/6J mice DCs and syngeneic C1498 mCh/luc + AML cells or C57BL/KaLwRij and syngeneic 5TGM1 mCh/luc + MM cells, respectively. T cells isolated from spleen were co-cultured with autologous irradiated DC/tumor fusions in presence of IL-2/7/15. Vaccine-educated T cells underwent selection with agonistic 4-1bb (3H3) followed by expansion with anti-CD3/CD28 activation beads. T cells were phenotyped for markers of activation (CD25/CD69), immune checkpoint (PD1/LAG3/TIM3), and memory (CD44+CD62L-) and for enrichment (anti-rat H&L). Cytotoxicity was evaluated on luminescent assay. Mice engrafted with C1498 mCh/luc + received the T cell therapy 7 days later. Disease progression and survival was monitored for 100 days by BLI. Results: In the C1498 model, vaccine-educated T cells demonstrated evidence of immune activation (CD25+CD69+) and memory (CD44+CD62L-) phenotype compared to unstimulated naïve T-cell controls (TN) (4-fold, p=0.1143; 1.3-fold, p=0.0195, respectively). Vaccine-educated T cells selected based on 4-1bb expression showed 9-fold higher expression of activation markers (p=0.0034) while memory markers were 4.4-fold higher compared to TN (p=0.0274). Selection enriched for 4-1bb + vaccine-educated T cells resulting in enhanced antigen-specific recognition as measured by induction of IFNg expression. Tumor specificity and activation was maintained following CD3/CD28-mediated expansion. The 4-1bb positive vaccine-educated T cells showed 11-fold enhanced cytotoxicity compared to TN at 10:1 E:T (P=0.0005), while vaccine-educated T cells showed 4-fold increase (P=0.0226). Phenotypic and functional analysis support 4 days as the optimal duration of time for T-cell vaccine education. Cell fold-expansion supports 3-5 days as the optimal duration for anti-CD3/CD28 expansion. In vivo, 60% of mice treated with 4-1bb + vaccine-educated cells were alive at 60 days vs 20% treated with unselected vaccine-educated cells. In the 5TGM1 model, activation, memory, immune checkpoint phenotype and cytotoxicity were increased in vaccine-educated T cells selected for 4-1bb expression. Conclusion: Vaccine-educated T cells represent a unique platform for adoptive immunotherapy for AML further enhanced by selection for 4-1bb expression by agonistic antibody. T-cell stimulation by fusion vaccine and subsequent enrichment by agonistic 4-1bb selection enhances cytotoxicity, activation and memory cell phenotype. Thus, ex vivo vaccine stimulation and 4-1bb selection presents a novel approach for cell-based immunotherapy for AML.

Large scale ex vivo expansion of clinical‑grade effector cells for adoptive immunotherapy

Cell-based adoptive immunotherapy for the treatment of various cancer types has attracted the attention of scientists. However, due to the absence of unitary standard protocols to produce large quantities of clinical-grade effector cells, it remains challenging to translate the experimental findings into clinical applications. The present study used methods complying with good manufacturing practice to induce effector cells from human peripheral blood mononuclear cells (PBMCs) of healthy donors by interleukin-2 and anti-Her-2 antibody with or without anti-CD3 antibodies (OKT3). The results indicated that the addition of OKT3 resulted in a greater expansion of the total cells and CD8+ T cells, and primarily induced the PBMCs to differentiate into CD3+ T cells. Regardless of the presence of OKT3, the expression of activating receptor of natural killer (NK) group 2, member D, and the inhibitory receptors of CD158a and CD158b on NK cells and NKT cells was increased, while the expression of NKp46 was inhibited on NK cells, but not on NKT cells. Furthermore, OKT3 did not affect the toxicity of the effector cells. Subgroup analysis indicated that although a variation of the composition of effector cells was present in different individuals under identical culture conditions, consistent marker expression on effector cells and target cell-killing effects were observed in different subgroups treated with or without OKT3. Furthermore, western blot analysis indicated that OKT3, apart from its involvement in cell cycle regulation, affects transcription and protein translation during processes of proliferation and differentiation. The present study provided experimental data regarding the production of effector cells for adoptive immunotherapy as a clinical application.

Cytokine-induced killer cells engineered with exogenous T-cell receptors directed against melanoma antigens: enhanced efficacy of effector cells endowed with a double mechanism of tumor recognition.

Cytokine-induced killer (CIK) cells consist of a heterogeneous population of polyclonal T lymphocytes displaying NK phenotype and HLA-unrestricted cytotoxic activity against a broad range of tumors. We sought to determine whether transduction of CIK cells with T cell receptor (TCR) genes specific for tumor-associated antigens could generate effector cells endowed with a double mechanism of tumor recognition. HLA-A2-restricted TCR-transduced (TD) CIK directed against the melanoma antigens Mart1 and NY-ESO1 were generated by lentiviral transduction and successfully expanded over a 3-4-week period. TD-CIK cells were both CD3(+)/CD56(-) and CD3(+)/CD56(+) (31±8% and 59±9%, respectively), indicating that both major histocompatibility complex (MHC)-restricted T cells and MHC-unrestricted CIK could be targeted by lentiviral transduction. At the end of the culture, the majority of both unmodified and TD-CIK displayed an effector memory phenotype, without considerable expression of replicative senescence and exhaustion markers. Functionally, TD-CIK specifically recognized tumor cells expressing the relevant antigen as well as maintained their MHC-unrestricted tumor activity. The cytotoxic activity of TD-CIK against HLA-A2(+) melanoma cell lines was significantly higher than the untransduced counterparts at a low effector:target ratio (cytotoxic activity of TD-CIK was from 1.9- to 4.3-fold higher than untransduced counterparts). TD-CIK were highly proficient in releasing high amount of IFN-γ upon antigen-specific stimulation and were able to recognize primary melanoma targets. In conclusion, we showed that (1) the reproducibility and simplicity of CIK transduction and expansion might solve the problem of obtaining adequate numbers of potent antitumor effector cells for adoptive immunotherapy; (2) the presence of both terminal effectors as well as of less differentiated progenitors might confer them long survival in vivo; and (3) the addition of an MHC-restricted antigen recognition allows not only targeting tumor surface antigens but also a wider range of cytoplasmic or nuclear antigens, involved in tumor proliferation and survival. TD-CIK cells with a double mechanism of tumor recognition are an attractive and alternative tool for the development of efficient cell therapeutic strategies.

Abstract 487: Effector B cells in cancer adoptive immunotherapy.

Abstract Successful anti-cancer treatment will have to appropriately stimulate both humoral as well as cellular immunity. To test this hypothesis, we reported that simultaneous targeting of CD3 on T cells and CD40 on B cells augmented the antitumor reactivity of tumor-primed LN cells. These studies established a role for engaging CD40 on tumor-draining lymph node (TDLN) B cells in the generation of effector cells. We also reported that IL-21 augmented the efficacy of T cell therapy by eliciting concurrent cellular and humoral responses. This study confirmed an interactive role between tumor-specific humoral responses related to IL-21 administration and adoptively transferred effector T cells. In recent study, we identified TDLN B cells as effector cells in an adoptive immunotherapy model. In vivo primed and in vitro activated TDLN B cells alone mediated effective (p<0.05) tumor regression after adoptive transfer into two histologically distinct murine tumor models. B cell plus T cell transfers resulted in substantially more efficient antitumor responses than B cells or T cells alone (p<0.05). Activated TDLN B cells produced IgM, IgG and IgG2b, which bound specifically to tumor cells and led to specific tumor cell lysis in the presence of complement. In a third tumor model, the 4T1 breast cancer spontaneous metastases model, we found that adoptive transfer of activated 4T1 TDLN B cells alone mediated significant inhibition of spontaneous metastases of the 4T1 cells from the injection site (the mammary fat pad) to the lung. Examination of the host revealed that the adoptive transfer of these B cells resulted in the induction of tumor specific T cell immunity as measured by cytotoxicity and cytokine (IFNγ and GM-CSF) production. Importantly, we found that the 4T1 TDLN effector B cells could directly and specifically kill the 4T1 tumor cells in the absence of antibody. More mechanistic studies revealed that adoptively transferred IL-10−/- TDLN B cells mediated significantly more effective antitumor immunity than equal numbers of WT TDLN B cells (p<0.05). Adoptively transferred IL-10−/− 4T1TDLN B cells increased the production of IgG which bound to 4T1 tumor cells and significantly increased CTL activity mediated by host B cells as well as T cells in an immunologically specific fashion. While the role played by B cells in the host immune response to cancer is complex and controversial, our results indicate that in vivo primed and in vitro activated TDLN B cells can function as effector cells in cancer adoptive immunotherapy, and removal of IL-10-producing B cell subsets may represent an effective strategy to augment the therapeutic efficacy of the TDLN effector B cells in cancer adoptive immunotherapy. Citation Format: Huimin Tao, Lin Lu, Martin Egenti, Alfred E. Chang, Qiao Li. Effector B cells in cancer adoptive immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 487. doi:10.1158/1538-7445.AM2013-487

Human effector CD8+ T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy

AbstractCluster of differentiation (CD)8+ T cells exist as naive, central memory, and effector memory subsets, and any of these populations can be genetically engineered into tumor-reactive effector cells for adoptive immunotherapy. However, the optimal subset from which to derive effector CD8+ T cells for patient treatments is controversial and understudied. We investigated human CD8+ T cells and found that naive cells were not only the most abundant subset but also the population most capable of in vitro expansion and T-cell receptor transgene expression. Despite increased expansion, naive-derived cells displayed minimal effector differentiation, a quality associated with greater efficacy after cell infusion. Similarly, the markers of terminal differentiation, killer cell lectin-like receptor G1 and CD57, were expressed at lower levels in cells of naive origin. Finally, naive-derived effector cells expressed higher CD27 and retained longer telomeres, characteristics that suggest greater proliferative potential and that have been linked to greater efficacy in clinical trials. Thus, these data suggest that naive cells resist terminal differentiation, or “exhaustion,” maintain high replicative potential, and therefore may be the superior subset for use in adoptive immunotherapy.

Targeting of G(D2)-positive tumor cells by human T lymphocytes engineered to express chimeric T-cell receptor genes.

Genetic engineering of human T lymphocytes to express tumor antigen-specific chimeric immune receptors is an attractive means for providing large numbers of effector cells for adoptive immunotherapy while bypassing major mechanisms of tumor escape from immune recognition. We have applied this strategy to the targeting of a G(D2)-positive tumor, neuroblastoma, which is the commonest extracranial solid tumor of childhood. Chimeric immune receptors were generated by joining an extracellular antigen-binding domain derived from either of the 2 ganglioside G(D2)-specific antibodies sc7A4 and sc14.G2a to a cytoplasmic signaling domain. The variable domains of hybridoma antibody 14.G2a were cloned and selected using a phage display approach. Upon coincubation with G(D2)-expressing tumor cell targets, human T lymphocytes transduced with recombinant retroviruses encoding chimeric receptors based on sc14.G2a, but not sc7A4, secreted significant levels of cytokines in a pattern comparable to the cytokine response obtained by engagement of the CD3 receptor. T cells transduced with the sc14.G2a-based chimeric T-cell receptors also displayed specific lysis of G(D2)-positive neuroblastoma cells, which was blocked in the presence of monoclonal antibody 14.G2a. In the absence of nonspecific stimulation of transduced cells, their functionality declined over time and antigenic stimulation of the chimeric receptor alone did not induce commitment to proliferation. These results support the feasibility of redirecting human T lymphocytes to a tumor-associated ganglioside epitope but emphasize that successful chimeric receptor-mediated adoptive immunotherapy will require additional strategies that overcome functional inactivation of gene-modified primary T lymphocytes.

Tumor site-selective localization of an adoptively transferred T cell line expressing a macrophage lectin.

CTLL-2 cells were transfected with an expression vector that contained cDNA of a mouse macrophage galactose/N-acetylgalactosamine-specific calcium-type lectin (MMGL) and a stable transfectant (CTL-ML) was established. These cells and mock transfectant cells (CTL-CEP) were labeled with a long-term fluorescent cell tracer, DiI. The labeled cells were intravenously injected into mice that contained established lung metastases produced by OV2944-HM-1 ovarian tumor cells. Analyses with fluorescence microscopy of a series of frozen lung sections from the recipient mice revealed that CTL-ML preferentially accumulated in the lung metastatic nodules, whereas CTL-CEP did not. Time course studies showed that the preferential accumulation was evident 3 days after adoptive transfer. We also found that OV2944-HM-1 cells bound peanut agglutinin and Vicia villosa agglutinin B4, whose sugar specificity overlaps with the specificity of MMGL. These results suggested that MMGL molecules expressed on CTLL-2 cells contributed to their selective trafficking or retention in tumor foci possibly through recognition of tumor-associated cell surface carbohydrate antigens. These results also suggested that MMGL could be used for the selective targeting of effector cells in adoptive immunotherapy.

Tissue distribution and tumor localization of effector cells in adoptive immunotherapy of cancer

In adoptive immunotherapy (AIT) of cancer, lymphocytes are isolated from the patient's blood and activated in vitro by the cytokine interleukin-2 (IL-2). In response to the IL-2 the lymphocytes proliferate vigorously and their cytotoxic potential increases several fold. After 5-10 days in culture, the cells-now called lymphokine-activated killer (LAK) cells-are injected back into the patient together with IL-2. The many positive results from preclinical animal models justified the rapid transit of AIT into the clinic, but the clinical results have far from fulfilled expectations. Many cancer centers have concluded that AIT in its present configuration is not cost-effective given that the average response rate is as low as 20-30%. Since a significant group of patients has shown complete responses after AIT, the challenge is to elucidate the conditions leading to optimal efficacy of AIT. It is generally accepted that the antineoplastic effect of LAK cells requires a close contact between the LAK cells and tumor cells. A central question in analyses of the mechanisms behind AIT is the ability of the LAK cells to localize to the malignant tissues. The earliest studies of the tissue distribution of 51Cr- and 111In-labeled LAK cells indicated that LAK cells, upon intravenous (i.v.) injection, are initially retained in the lungs, but redistribute to liver and spleen during the following 16-24 hours. However, our studies of the traffic and fate of i.v. injected tumor cells have shown that the use of 51Cr and 111In as cell labels often results in an over-estimation of the traffic of cells to liver and spleen and leads to falsely high predictions as to the survival of the injected cells, due to non-specific accumulation of 51Cr and 111In in liver and spleen after their release from dead cells. Use of 125IUdR, which does not accumulate in liver and spleen following release from dead cells, shows that the traffic of LAK cells into these organs was much lower than previously thought. These experiments have now been repeated using other cell labels (such as fluorescence dyes and immunohistochemistry) and they confirm that only few LAK cells redistribute from the lungs to the liver and spleen and that most die within the first 24 hours following injection. Thus, the circulatory potential of LAK cells is very low and chances that i.v. injected LAK cells will be able to localize into tumors and metastases located in other organs than the lungs, seems small. Indeed, while fluorescence-labeled LAK cells selectively localize into pulmonary metastases following intravenous injection, no infiltration of extrapulmonary metastases is seen. Furthermore, quantitative analyses have shown that even though the localization of LAK cells into pulmonary metastases is highly specific (5-10 fold higher numbers of LAK cells are often found in the metastases compared to the surrounding normal lung tissue), only 5% of the injected cells reach the malignant tissues. It is therefore reasonable to assume that the efficacy of AIT can be improved if the in vivo survival of the LAK cells can be prolonged and if their ability to infiltrate tumors regardless of their location, can be augmented. Previous studies in murine models have shown that i.v. injected tumor cells are sequestrated in the lungs and that only few of them reach other organs. However, when the tumor cells were injected into the left ventricle of the heart (bypassing the lung capillaries), significant numbers of tumor cells were found in the liver. It therefore seemed reasonable to speculate that LAK cells injected into the left ventricle of the heart or directly into the arteries supplying the tumor-bearing organ would have better chances of localizing to the malignant tissue. This seemed to be correct in that 10 fold higher numbers of LAK cells were found in the liver following intraportal injection compared to intravenous injection.

Induction in vitro of a primary human antiviral cytotoxic T cell response

We report herein the successful priming of human anti-viral cytotoxic T cells (CTL) in vitro using two induction strategies based on the stimulation of peripheral blood mononuclear cells isolated from uninfected donors with synthetic viral peptides. The peptides used contain HLA-A2 binding motifs and have been identified as HLA-A2-restricted CTL epitopes in patients infected by the hepatitis B and C viruses. One approach uses repetitive long-term stimulation and the other uses bulk cultures containing large numbers of naive peripheral blood mononuclear cells. Both approaches successfully induce HLA-A2-restricted CTL specific for several viral epitopes. Some CTL recognize endogenously synthesized antigen on target cells infected with recombinant vaccinia virus expressing the corresponding viral proteins. This simple technique permits easy analysis of the primary human CTL repertoire, and may be exploitable for production of specific CTL effector cells for adoptive immunotherapy and dissection of the cellular and molecular requirements for priming of naive human CTL.

Generation of T-Cells Reactive to the Poorly Immunogenic B16-BL6 Melanoma with Efficacy in the Treatment of Spontaneous Metastases

The B16-BL6 (BL6) melanoma is a poorly immunogenic murine tumor that is highly invasive and spontaneously metastasizes from the primary site. Utilizing an established anti-CD3/interleukin-2 (IL-2) culture procedure, we have previously reported that lymph nodes (LNs) draining immunogenic murine sarcomas contained preeffector cells that could be activated to differentiate into therapeutic effector cells for adoptive immunotherapy. By contrast, LNs draining the poorly immunogenic BL6 melanoma were found not to be a reliable source of preeffector cells. Instead, sensitization of preeffector cells reactive to BL6 required the subcutaneous inoculation of tumor admixed with Corynebacterium parvum. LN cells draining these vaccination sites demonstrated therapeutic efficacy only after subsequent anti-CD3/IL-2 activation. The sensitization of preeffector cells was dependent on the presence of tumor antigen and an optimal dose of C. parvum (< or = 50 micrograms). Furthermore, kinetic analysis revealed that the preeffector response was transient after tumor vaccination. The therapeutic efficacy of anti-CD3/IL-2 activated LN cells was further evaluated in the treatment of spontaneous macroscopic BL6 visceral metastases. Spontaneous visceral metastases were induced in animals by inoculation with BL6 tumor in the footpad followed by amputation of the primary tumor 3 weeks later. The systemic transfer of 10(8) anti-CD3/IL-2 activated T-cells and the concomitant intraperitoneal administration of subtherapeutic doses of IL-2 1 week after amputation cured 50% of the animals and prolonged median survival time (MST) to > 140 days. All mice except one that received no treatment or was treated with IL-2 alone succumbed to visceral metastases with an MST of approximately 23 days. This study characterizes a model whereby the weak immune response to the BL6 melanoma can be positively or negatively modulated for the generation of antitumor reactive T-cells useful in adoptive immunotherapy.

口腔悪性腫よう組織浸潤リンパ球からの抗腫よう活性の誘導に関する研究

Previous reports indicated that the histological intensity of the tumor infiltrating lymphocytes (TIL), which is one of the manifestations of immunological host's response against malignant tumor cells, correlates with the clinical prognosis of the cancer patients.In the present study, we investigated the antitumor cytotoxicity of TIL (or LN-TIL: lymphocytes of metastatic lymph node) induced by anti-CD3 antibody and interleukin 2 (IL-2), and the availability for the source of the adoptive immunotherapy on patients with oral malignant tumors.TIL were isolated from 28 tumors and LN-TIL were isolated from 6 metastatic lymph nodes obtained from 28 patients with oral malignant tumors.Natural killer (NK) and lymphokine-activated killer (LAK) activity of freshly isolated TIL were usually undetectable. However the antitumor cytotoxicity of TIL was generated with IL-2 in vitro culture. These activated TIL showed high levels of NK and LAK activities, and these were prolonged more than those of PBL.On the other hand, freshly isolated TIL from patients who received IL-2 injection directly into the tumor lesions before surgery, showed NK and LAK activities.The growth of TIL were obviously enhanced by using the plates coated with anti-CD3 antibody at the beginning of the culture.These activated TIL (or LN-TIL) with anti-CD3 antibody and/or IL-2 also showed the cytotoxicity against the autologous tumor cells.These results indicate that anti-CD3 antibody and IL-2 augment the specific and/or nonspecific cytotoxicity of the TIL (or LN-TIL) against the tumor cells, and this augmentation of cytotoxic activity shows the anti-tumoral efficacy for oral malignant tumors. Furthermore, TIL (or LN-TIL) from oral malignant tumors could be on appropriate effector cells for adoptive immunotherapy on the patients with oral malignant tumors.

Characterization of anti-CD8-resistant cytolytic T lymphocytes induced by multivalent cross-linking of CD8 on precursor cells.

The lytic activity of most CD8+ MHC class I allospecific CTL generated in vitro can be inhibited by anti-CD8 antibodies. Such inhibition has led to hypotheses that CD8/class I interactions normally contribute to the triggering of CTL with low or moderate avidity Ag-specific TCR by providing those CTL with auxiliary binding avidity. However, CD8 has also been proposed to play an active signaling role in T cell activation. We have recently reported that multivalent cross-linking of CD8 on CTL precursors in MLC does appear to mediate activation signals, and induces the generation of CD8+ MHC class I allospecific CTL whose lytic activity cannot be blocked by anti-CD8 antibodies. In our present study, we have further characterized such anti-CD8 uninhibitable effector cells. These CTL are resistant to blocking of their lytic function by anti-Lyt-3 mAb as well as anti-Lyt-2 mAb, but remain sensitive to blocking by anti-LFA-1 mAb, indicating that they do use non-CD8 cell adhesion molecules during target cell recognition and lysis. As a consequence of mAb-induced multivalent CD8 cross-linking during their generation, anti-CD8 uninhibitable CTL significantly reduce their cell surface expression of CD8, which permits their identification and facilitates their purification from heterogeneous MLC populations. Such anti-CD8 uninhibitable effector cells can be maintained as stable CTL lines, in the absence of anti-CD8 mAb after the initial induction period. The in vitro generation of anti-CD8 uninhibitable CTL, which may be highly enriched for cells bearing high affinity TCR, could represent a new experimental approach to studies of TCR gene usage and repertoire, as well as a potentially important strategy for the deliberate generation of high affinity effector cells for adoptive immunotherapy.

Natural cell-mediated cytotoxicity in vitro and inhibition of tumor growth in vivo by murine lymphoid cells cultured with T cell growth factor (TCGF).

Lymphoid cells obtained from the spleen, thymus, bone marrow, peripheral blood, and peritoneal exudate of normal mice (BALB/c, BALB/c nude, C57BL/6, C3H) and from spleens of mice bearing a transplantable lung carcinoma or primary mammary carcinoma were expanded in culture for 1-9 months, with an increase in cell number of 10(5)- to 10(6)-fold per month, in crude or lectin-depleted medium containing T cell growth factor (TCGF). All these cultured lymphoid cell (CLC) lines exhibited strong cytotoxic activity in vitro (assessed by 51Cr-release assays) toward a variety of freshly harvested and cultured syngeneic, allogeneic, and xenogeneic tumor target cells, both lymphoid and solid (including metastatic growths) in origin. Extensive killing was observed against tumor targets that were resistant to lysis by natural killer (NK) cells as well as to NK-sensitive tumor lines. Low levels of cytotoxic reactivity were also demonstrated against fresh and cultured normal lymphoid cells. The CLC had some characteristics of NK cells but also expressed some typical T cell markers. In local Winn-type neutralization assays, CLC delayed or completely inhibited the growth of lymphomas and carcinomas in syngeneic and allogeneic recipients. In mice with metastatic growth of a second-generation transplant of mammary carcinoma, CLC were shown to have some therapeutic effect when administered IV 1 day after cyclophosphamide. No significant beneficial action of IV administered CLC was observed in the absence of chemotherapy in mice implanted with a lung carcinoma. The possibilities of employing TCGF-propagated cytotoxic effector cells in adoptive immunotherapy of human malignancies are discussed.