405. IL-2 and TNF-a Coding Adenoviruses Enable Adoptive T Cell Therapy in Metastatic, Solid Cancer by Systemically Activating Tumor-Reactive TILs
Siri TähtinenC BlattnerMarkus Vähä‐KoskelaDipongkor SahaMikko SiuralaRiikka HavunenSuvi ParviainenJochen UtikalViktor UmanskyAkseli Hemminki
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Adoptive T cell therapy (ACT) using genetically modified T cells has shown exceptional efficacy in the treatment of CD19+ hematological cancers. However, far less impressive results have been achieved in the treatment of solid tumors due to local immunosuppression, rendering tumor-infiltrating T cells (TILs) hypofunctional. We have previously shown that adenovirus (Ad) infection can enhance the efficacy of ACT (Tähtinen et al, CIR 2015) and that intratumoral administration of immunostimulatory cytokines can result in favorable alteration of tumor microenvironment (Tähtinen et al, PLOS One 2015). To combine the benefits of both approaches, we studied if replication-deficient Ad5-vectors coding for interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-a) would affect the activity of adoptively transferred, TCR-transgenic TRP-2(180-188) specific T cells in vivo. To gain clinically relevant mechanism-of-action data, we chose to use ret transgenic mouse model that develops spontaneous malignant skin melanoma which metastasizes into distant organs. Following ACT and intratumoral virus injection, a significant increase in activated PD-1+ CD8+ T cells was seen in both cutaneous lesions and in metastatic lymph nodes. Interestingly, a reverse correlation between tumor weight and the number of tumor-reactive PD-1+ TILs (p=0.0015) was observed, indicating that T-cell hypofunction was overcome and successful tumor lysis was achieved. Local expression of cytokines did not affect the levels of immunosuppressive immune cell subsets such as myeloid-derived suppressor cells (MDSCs) or T regulatory cells (Tregs), latter of which has previously been associated with systemic IL-2 therapy (Ahmadzadeh and Rosenberg, Blood 2006). Instead, Ad5-IL2 treatment induced upregulation of IL-2 receptor α-chain (CD25) in conventional CD4+CD25+Foxp3-cells, suggesting that these helper T cells contributed to CD8+ TIL activation. Finally, beneficial ratios between tumor-reactive PD-1+ CD8+ TILs and Tregs was observed in primary and secondary tumor sites, indicating that IL-2 and TNF-a coding adenoviruses can modify the cellular composition of the tumor microenvironment in favor of adoptively transferred T cells. In conclusion, IL-2 and TNF-a coding adenoviruses can break tumor-associated immunotolerance and significantly increase the levels of active, tumor-reactive T-cells both in injected cutaneous lesions and in non-injected metastatic lymph nodes. Importantly, this triple modality may represent an efficient approach to achieve "CD19-like" clinical responses in the treatment of solid, metastatic cancers currently incurable by standard therapies.Keywords:
Adoptive Cell Transfer
Abstract KCl extracts of Melanoma 14, a human melanoma cell line grown in chemically defined serum‐free medium, inhibited leukocyte migration in 19/36 (53%) patients with malignant melanoma. Only 4/23 (17%) controls with non‐melanoma malignancies and 4/28 (14%) normal subjects with no history of cancer were similarly inhibited. Only 2/27 melanoma patients tested against KCl extracts of normal muscle tissue excised from the donor of Melanoma 14 were significantly inhibited. Patients with Stage I (localized) melanoma and patients with Stage III (generalized) melanoma reacted with roughly equal frequency but the number of patients in each group was too small for meaningful statistical analysis. Leukocytes from the donor of Melanoma 14 were tested in a completely autologous system against extracts of Melanoma 14 tissue culture cells and extracts of autologous muscle and were specifically inhibited by the Melanoma 14 tissue culture extract (Migration Index = 0.67) but not by the extract of normal muscle (Migration Index = 0.96). Only 7/32 (22%) melanoma patients were significantly inhibited by an extract of non‐melanoma tumor. These results suggest that melanoma‐associated antigens are present in soluble extracts of this tumor line. Such extracts could provide a continuing source of standard melanoma‐associated antigens for purification and chemical characterization and for diagnostic and prognostic tests in patients with malignant melanoma.
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In a mixed leukocyte culture (MLC) reaction of allogenic mouse spleen cells differing for H-2K or H-2D, only a weak cytotoxic response is generated. This cytotoxic response is augmented significantly if bacterial lipopolysaccharide (LPS), 5 microgram/ml, or polyadenylic acid (poly A):polyuridylic acid (poly U), 20 microgram/ml, is present in the culture. The cytotoxic cells generated in the presence of these two agents are specific for sensitizing H-2K or H-2D antigen. Two lines of evidence suggest that these two agents exert their effect at different steps in the development of cytotoxic lymphocytes: (a) the effect of poly A:U depends on the presence of adherent cells, whereas the effect of LPS is independent of the presence of adherent cells and (b) LPS promotes the development of cytotoxic cells when ultraviolet light-treated stimulating cells are used in the MLC whereas poly A:U does not.
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Abstract The main conclusion from these experiments is that the antigen‐specific suppressor T cell of mice which inhibits the induction of cytotoxic T lymphocytes is not itself a cytotoxic T cell. This conclusion is supported by two main observations: first, a certain cell number from first‐step cultures which was suppressive in the presence of a high dose of antigen actually helped the cytotoxic response at a lower antigen dose. This observation is difficult to reconcile with the hypothesis that suppression is due to the killing of the stimulator or the responder cells in the second‐step culture by cytotoxic T cells. Second, cells from first‐step cultures of cortisone‐treated mice displayed cytotoxic activity but had no suppressive effect on the generation of killer cells. It was further demonstrated that these cells failed to influence in any way the suppressive effect, however weak, of cells from first‐step cultures of normal spleen. We therefore favor the view that the suppression observed in this system is due to a regulatory signal which occurs as a result of the ability of both inhibitory cells and responder cells to recognize and respond to allogeneic determinants expressed on the surface of stimulator cells. The suppressor T cells described here act by linked associative recognition of antigen. That is, suppressor T cells only inhibit the induction of a precursor cytotoxic T cell in the presence of an antigen to which both the precursor cell and the suppressor cell can bind. In this sense, suppressors act in a manner analogous to helper T cells in T‐B cell cooperation; carrier‐specific helper T cells only enhance an anti‐hapten B cell response in the presence of hapten‐carrier conjugates. Similarly, alloantigen a (carrier)‐specific suppressor T cells only inhibit alloantigen b (hapten)‐specific cytotoxic responses in the presence of (a × b)F 1 stimulator cells (hapten‐carrier conjugate), not in the presence of a mixture of parental stimulator cells (a + b).
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Sir: A number of investigators have recently reported that cell membranes, or solubilized proteins derived from them, are capable of inducing cytotoxic responses in secondary cultures (1–6). They have implied thereby that they are gaining insight into the specificity of the structures recognized by cytotoxic T cells. An alternative interpretation seems equally plausible: that the membrane preparations act not directly on cytotoxic memory cells, but rather on helper T cells, which, by secreting soluble mediators, “trigger” cytotoxic cell differentiation. This interpretation is supported by several recent reports. Thus, Ryser et al. (7), Wagner and Rollinghoff (8), and this laboratory (Okada et al. , in press) have shown that cell mediators secreted by primed Lyt I+ T cells can cause the direct differentiation of cytotoxic T cells in secondary (memory) cultures. Moreover, the specificity of the cytotoxic cells induced by these mediators is that of the antigen initially used for priming.
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Abstract Sir: A number of investigators have recently reported that cell membranes, or solubilized proteins derived from them, are capable of inducing cytotoxic responses in secondary cultures (1–6). They have implied thereby that they are gaining insight into the specificity of the structures recognized by cytotoxic T cells. An alternative interpretation seems equally plausible: that the membrane preparations act not directly on cytotoxic memory cells, but rather on helper T cells, which, by secreting soluble mediators, “trigger” cytotoxic cell differentiation. This interpretation is supported by several recent reports. Thus, Ryser et al. (7), Wagner and Rollinghoff (8), and this laboratory (Okada et al., in press) have shown that cell mediators secreted by primed Lyt I+ T cells can cause the direct differentiation of cytotoxic T cells in secondary (memory) cultures. Moreover, the specificity of the cytotoxic cells induced by these mediators is that of the antigen initially used for priming.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces a T cell response that most likely contributes to virus control in COVID-19 patients but may also induce immunopathology. Until now, the cytotoxic T cell response has not been very well characterized in COVID-19 patients. Here, we analyzed the differentiation and cytotoxic profile of T cells in 30 cases of mild COVID-19 during acute infection. SARS-CoV-2 infection induced a cytotoxic response of CD8+ T cells, but not CD4+ T cells, characterized by the simultaneous production of granzyme A and B as well as perforin within different effector CD8+ T cell subsets. PD-1-expressing CD8+ T cells also produced cytotoxic molecules during acute infection, indicating that they were not functionally exhausted. However, in COVID-19 patients over the age of 80 years, the cytotoxic T cell potential was diminished, especially in effector memory and terminally differentiated effector CD8+ cells, showing that elderly patients have impaired cellular immunity against SARS-CoV-2. Our data provide valuable information about T cell responses in COVID-19 patients that may also have important implications for vaccine development.IMPORTANCE Cytotoxic T cells are responsible for the elimination of infected cells and are key players in the control of viruses. CD8+ T cells with an effector phenotype express cytotoxic molecules and are able to perform target cell killing. COVID-19 patients with a mild disease course were analyzed for the differentiation status and cytotoxic profile of CD8+ T cells. SARS-CoV-2 infection induced a vigorous cytotoxic CD8+ T cell response. However, this cytotoxic profile of T cells was not detected in COVID-19 patients over the age of 80 years. Thus, the absence of a cytotoxic response in elderly patients might be a possible reason for the more frequent severity of COVID-19 in this age group than in younger patients.
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Abstract Targeted disruption of β 2 ‐microgobulin gene results in deficient major histocompatibility complex class I expression and failure to develop CD4 − 8 + T cells. Despite this, β 2 M −/− mice reject skin grafts and cope with most viral infections tested. We asked whether CD4 + 8 − cytotoxic T cells could play a role in compensating for the defect in CD4 − 8 + cytotoxic T cell function. We found that the cytotoxic activity against class II + targets is significantly higher among CD4 + 8 − T cells of β 2 M −/− than among those of β 2 M +/+ mice. In the limiting dilution experiment, we showed that the precursor frequency for the cytotoxic, CD4 + 8 − , class II‐specific T cells is at least fivefold higher in β 2 M −/− than in β 2 M +/+ mice. These results suggest that CD4+8 − cytotoxic T cells could play a major role in carrying out cytotoxic function in β 2 M −/− mice.
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