Distinct and overlapping roles of interleukin-10 and CD25+ regulatory T cells in the inhibition of antitumor CD8 T-cell responses

Lack of antitumor immunity is often related to impaired CD8 T-cell responses that could result from a poor priming capacity by tumor-infiltrating dendritic cells (TIDC) and/or further inhibition by regulatory T cells (T(reg)). Interleukin-10 (IL-10) has been implicated in the inhibition of TIDC as well as in the generation and functions of T(reg). Here, we address some of the respective and possibly overlapping roles of IL-10 and CD25+ T(reg) in CD8 antitumor immunity. Whereas tumor antigen-specific CD8 T cells proliferated in vivo in the presence of IL-10 or T(reg), optimal effector functions were observed in mice lacking both IL-10 and T(reg). Indeed, tumors grown in normal but not in IL-10-deficient or CD25-depleted mice induced tumor antigen-specific CD8 suppressor T cells. Suppression involved transforming growth factor-beta. Similarly, both IL-10 and T(reg) were responsible for impaired CD8 T cell priming by TIDCs, but IL-12 production by TIDCs was prevented only by T(reg)-independent IL-10. Subsequently, IL-10 defect and T(reg) depletion were required to achieve optimal induction of CD8 T-cell effectors by TIDC following CpG activation. Our results point out major redundant and nonredundant roles for IL-10 and T(reg) in the inhibition of TIDC-mediated generation of antitumor CD8 T-cell response.     

In vivo plasmid electroporation induces tumor antigen-specific CD8+ T-cell responses and delays tumor growth in a syngeneic mouse melanoma model

Plasmid DNA-based molecular cancer vaccines generally suffer from suboptimal immunogenicity. One of the key limitations is insufficient level of gene expression, which was surmounted in our approach by using the novel technique of in vivo plasmid electroporation-enhanced vaccination (electrovaccination). Electrovaccination with plasmids encoding the full-length autologous melanocyte antigen tyrosinase-related protein-2 induced limited melanocyte destruction in a subset of mice. Despite examples of vitiligo, vaccinated mice were not protected from a subsequent challenge of B16F10M melanoma cells. Novel constructs were then designed and submitted to a functional screen. Best performance was obtained when the relevant H-2K(b)-restricted epitope SVYDFFVWL was placed into a context of sequences of the HLA-Cw3 molecule. After animals were electrovaccinated using this construct, direct enzyme-linked immunospot analysis of peripheral blood mononuclear cells indicated that very high numbers of T cells recognizing the specific tyrosinase-related protein-2 epitope were generated. CD8+ T cells isolated from the spleen also displayed a high degree of antigen-specific reactivity and vigorously reacted toward unmodified B16F10M cells. In vivo protective effects of this construct were demonstrated in mice using two different models; outgrowth of s.c. implanted B16F10M tumor cells was significantly delayed, and vaccinated mice developed no or only very few tumor nodules in an i.v. lung metastasis model. Thus, improved antigen vectors delivered by highly effective gene transfer methods may form the basis for future human applications.     

CD4+ T cells are able to promote tumor growth through inhibition of tumor-specific CD8+ T-cell responses in tumor-bearing hosts

Modulation of the immune response by established tumors may contribute to the limited success of therapeutic vaccination for the treatment of cancer compared with vaccination in a preventive setting. We analyzed the contribution of the CD4+ T-cell population to the induction or suppression of tumor-specific CD8+ T cells in a tumor model in which eradication of tumors crucially depends on CD8+ T cell-mediated immunity. Vaccine-mediated induction of protective antitumor immunity in the preventive setting (i.e., before tumor challenge) was CD4+ T cell dependent because depletion of this T-cell subset prevented CD8+ T-cell induction. In contrast, depletion of CD4+ cells in mice bearing established E1A+ tumors empowered the mice to raise strong CD8+ T-cell immunity capable of tumor eradication without the need for tumor-specific vaccination. Spontaneous eradication of tumors, which had initially grown out, was similarly observed in MHC class II-deficient mice, supporting the notion that the tumor-bearing mice harbor a class II MHC-restricted CD4+ T-cell subset capable of suppressing a tumor-specific CD8+ T-cell immune response. The deleterious effects of the presence of CD4+ T cells in tumor-bearing hosts could be overcome by CD40-triggering or injection of CpG. Together these results show that CD4+ T cells with a suppressive activity are rapidly induced following tumor development and that their suppressive effect can be overcome by agents that activate professional antigen-presenting cells. These observations are important for the development of immune interventions aiming at treatment of cancer.     

Combinational FLt3 ligand and granulocyte macrophage colony-stimulating factor treatment promotes enhanced tumor infiltration by dendritic cells and antitumor CD8(+) T-cell cross-priming but is ineffective as a therapy

Dendritic cells play significant roles in the development and maintenance of antitumor immune responses. Therapeutic recruitment of dendritic cells into the tumor microenvironment has the potential to result in enhanced antitumor T-cell cross-priming against a broad array of naturally processed and presented tumor-associated antigens. We have observed that the treatment of BALB/c mice bearing syngeneic CMS4 sarcomas with the combination of recombinant Flt3 ligand and recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) for five sequential days is sufficient to optimize the number of tumor-infiltrating dendritic cells (TIDC). However, despite the significant increase in the number of TIDCs, the therapeutic benefit of Flt3 ligand and GM-CSF treatment is minimal. Therapy-associated TIDCs do not exhibit a "suppressed" or "suppressor" phenotype in vitro, and their enhanced numbers in cytokine-treated mice were associated with increased levels of peripheral antitumor CD8(+) T effector cells and with an augmented population of CD8(+) tumor-infiltrating lymphocytes (TIL). These data suggest that Flt3 ligand + GM-CSF therapy of murine tumors fails at a mechanistic point that is downstream of specific T-cell priming by therapy-induced TIDCs and the recruitment of these T cells into the tumor microenvironment. Based on the enhanced infiltration of tumors by CD4(+)CD25(+) TIL in Flt3 ligand + GM-CSF-treated mice, this could reflect the dominant influence of regulatory T cells in situ.     

CD20 antibody (C2B8)-induced apoptosis of lymphoma cells promotes phagocytosis by dendritic cells and cross-priming of CD8+ cytotoxic T cells.

C2B8 (Rituximab, MabThera) is a chimeric mouse/human monoclonal antibody (mAb) directed against the human B cell-restricted cell surface antigen CD20 which is used as an alternative medication in the treatment of B cell non-Hodgkin lymphomas (NHL). Treatment of CD20+ B cells with C2B8 triggers different cell damaging effects including complement-dependent lysis of tumor cells, antibody-dependent cellular cytotoxicity and induction of apoptosis. Dendritic cells (DC) have recently been shown to ingest cell debris and to present associated antigens even on MHC class I molecules, a mechanism called cross-presentation. In this study, we investigated whether C2B8 treatment of lymphoma promotes the induction of CD8+ T cell responses against lymphoma cell-associated antigens via, cross-presentation. We used Daudi lymphoma cells as a model system in our studies and could demonstrate, that C2B8-treated Daudi cells undergo apoptosis, are phagocytosed by DC and induce in DC typical features of maturation; among them, the induction of CD83 expression as well as the up-regulation of prominent accessory molecules (CD40, CD86) and MHC molecules. Importantly, upon co-culture of such lymphoma cell-pulsed DC with autologous T cells, we could induce efficient cytotoxic T cell (CTL) responses against Daudi cell-associated antigens. These findings suggest that antibody treatment of tumor cells can, in addition to its direct cell damaging effects, under certain conditions, contribute to an induction of potentially protective cytotoxic T cell responses.     

A tumor lysate is an effective vaccine antigen for the stimulation of CD4+ T-cell function and subsequent induction of antitumor immunity mediated by CD8+ T cells

To develop a potent cancer vaccine, it is important to study how to prepare highly immunogenic antigens and to identify the most appropriate adjuvants for the antigens. Here we show that a tumor lysate works as an effective antigen to prime CD4⁺ T-cell help when baculovirus is employed as an adjuvant. When immunized intradermally with the combination (BLP) of baculovirus, a CT26 tumor lysate, and a cytotoxic T-cell epitope peptide before a tumor challenge, 60% of mice rejected tumors. In contrast, all mice vaccinated with baculovirus plus a tumor lysate (BL) developed tumors. In addition, flow cytometry showed that tumor-specific, interferon γ-producing CD8⁺ cytotoxic T lymphocytes (CTLs) were robustly activated by intradermal immunization with BLP. When BLP was administered therapeutically to tumor-bearing mice, antitumor efficacy was better compared to BL. The established tumor was completely eradicated in 50–60% of BLP-treated mice, and induction of tumor-specific CTLs was observed, suggesting that the antitumor efficacy of BLP is mediated by CD8⁺ T cells. Numerous CD4⁺ T cells infiltrated the tumors of BLP-treated mice, whereas the antitumor effect of BLP almost disappeared after removal of the tumor lysate from BLP or after depletion of BLP-immunized mice of CD4⁺ T cells. Thus, the combination of a peptide, lysate, and baculovirus provides stronger antitumor immunity than does a peptide plus baculovirus or a lysate plus baculovirus; effectiveness of BLP is determined by functioning of CD4⁺ T cells stimulated with a tumor lysate.     

Maximal T cell-mediated antitumor responses rely upon CCR5 expression in both CD4(+) and CD8(+) T cells

Immune responses against cancer rely upon leukocyte trafficking patterns that are coordinated by chemokines. CCR5, the receptor for chemotactic chemokines MIP1alpha, MIP1beta, and RANTES (CCL3, CCL4, CCL5), exerts major regulatory effects on CD4(+)- and CD8(+) T cell-mediated immunity. Although CCR5 and its ligands participate in the response to various pathogens, its relevance to tumoral immune control has been debated. Here, we report that CCR5 has a specific, ligand-dependent role in optimizing antitumor responses. In adoptive transfer studies, efficient tumor rejection required CCR5 expression by both CD4(+) and CD8(+) T cells. CCR5 activation in CD4(+) cells resulted in CD40L upregulation, leading to full maturation of antigen-presenting cells and enhanced CD8(+) T-cell crosspriming and tumor infiltration. CCR5 reduced chemical-induced fibrosarcoma incidence and growth, but did not affect the onset or progression of spontaneous breast cancers in tolerogenic Tg(MMTV-neu) mice. However, CCR5 was required for TLR9-mediated reactivation of antineu responses in these mice. Our results indicate that CCR5 boosts T-cell responses to tumors by modulating helper-dependent CD8(+) T-cell activation.     

Immune surveillance: both CD3+ CD4+ and CD3+ CD8+ T cells control in vivo growth of P815 mastocytoma.

The aim of this study was to examine whether a spontaneous immune response controls neoplastic growth in P815-bearing DBA/2 mice, and to characterize the cells involved in tumor resistance in vivo. Several cell lineages such as T-cell-receptor (TcR)-bearing T cells, NK cells and macrophages mediate some anti-tumor activity in vitro. P815 was chosen as a model because it is weakly immunogenic and is a good target both for tumor-specific, MHC-restricted CTL-mediated lysis and for MHC-unrestricted lysis exerted by long-term cultured lymphocytes or activated macrophages. Since most "NK-like activity" in freshly isolated populations appears to be associated with CD3- cells, whereas antigen-specific, MHC-restricted T cells mostly express CD3 determinants, CD3 was a good marker for evaluating the role of T cells and "NK" cells in tumor resistance in vivo. The survival of anti-CD3-treated animals that were inoculated with tumor cells was strongly reduced (mean survival time: 17 days vs. 40 days for the control group) and was associated with increased tumor growth rate. We followed the same approach to define the T-cell subset(s) that mediate(s) this immune response. Both CD4+ and CD8+ T cells were required for induction of immune control on neoplastic growth. The approach used has revealed the important role of CD4+ T cells in immune responses that control in vivo growth of a class-I-positive, class-II-negative tumor and suggests that these cells may play a central role in tumor resistance. Since CD4+ cells are activated by soluble, exogenous proteins, this finding may have important implications for immunotherapy.     

Dissociation of its opposing immunologic effects is critical for the optimization of antitumor CD8+ T-cell responses induced by interleukin 21

PURPOSE: Interleukin 21 (IL-21) is a promising new cytokine, which is undergoing clinical testing as an anticancer agent. Although IL-21 provides potent stimulation of CD8(+) T cells, it has also been suggested that IL-21 is immunosuppressive by counteracting the maturation of dendritic cells. The dissociation of these two opposing effects may enhance the utility of IL-21 as an immunotherapeutic. In this study, we used a cell-based artificial antigen-presenting cell (aAPC) lacking a functional IL-21 receptor (IL-21R) to investigate the immunostimulatory properties of IL-21. EXPERIMENTAL DESIGN: The immunosuppressive activity of IL-21 was studied using human IL-21R(+) dendritic cells. Antigen-specific CD8(+) T cells stimulated with human cell-based IL-21R(-)aAPC were used to isolate the T-cell immunostimulatory effects of IL-21. The functional outcomes, including phenotype, cytokine production, proliferation, and cytotoxicity were evaluated. RESULTS: IL-21 limits the immune response by maintaining immunologically immature dendritic cells. However, stimulation of CD8(+) T cells with IL-21R(-) aAPC, which secrete IL-21, results in significant expansion. Although priming in the presence of IL-21 temporarily modulated the T-cell phenotype, chronic stimulation abrogated these differences. Importantly, exposure to IL-21 during restimulation promoted the enrichment and expansion of antigen-specific CD8(+) T cells that maintained IL-2 secretion and gained enhanced IFN-gamma secretion. Tumor antigen-specific CTL generated in the presence of IL-21 recognized tumor cells efficiently, demonstrating potent effector functions. CONCLUSIONS: IL-21 induces opposing effects on antigen-presenting cells and CD8(+) T cells. Strategic application of IL-21 is required to induce optimal clinical effects and may enable the generation of large numbers of highly avid tumor-specific CTL for adoptive immunotherapy.     

Increased CD8+ T-cell function following castration and immunization is countered by parallel expansion of regulatory T cells

Although androgen ablation therapy is effective in treating primary prostate cancers, a significant number of patients develop incurable castration-resistant disease. Recent studies have suggested a potential synergy between vaccination and androgen ablation, yet the enhanced T-cell function is transient. Using a defined tumor antigen model, UV-8101-RE, we found that concomitant castration significantly increased the frequency and function of antigen-specific CD8(+) T cells early after the immunization of wild-type mice. However, at a late time point after immunization, effector function was reduced to the same level as noncastrated mice and was accompanied by a concomitant amplification in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) following immunization. We investigated whether Treg expansion occurred following castration of prostate tumor-bearing mice. In the prostate-specific Pten(-/-) mouse model of prostate cancer, we observed an accelerated Treg expansion in mice bearing the castration-resistant endogenous prostate tumor, which prevented effector responses to UV-8101-RE. Treg depletion together with castration elicited a strong CD8(+) T-cell response to UV-8101-RE in Pten(-/-) mice and rescued effector function in castrated and immunized wild-type mice. In addition, Treg expansion in Pten(-/-) mice was prevented by in vivo interleukin (IL)-2 blockade suggesting that increased IL-2 generated by castration and immunization promotes Treg expansion. Our findings therefore suggest that although effector responses are augmented by castration, the concomitant expansion of Tregs is one mechanism responsible for only transient immune potentiation after androgen ablation.     

前列腺素E2对肿瘤微环境中髓源性抑制细胞的调控作用

髓源性抑制细胞(M DSC)是一群未成熟的骨髓异质性细胞,在肿瘤免疫耐受和免疫抑制中具有重要作用.近年越来越多的研究结果表明前列腺素E2(PGE2)与MDSC有着密不可分的关系,PGE2在肿瘤微环境中通过作用其相关受体,调控信号转导与转录激活因子3(STAT3)和蛋白激酶A等细胞信号通路及细胞因子的分泌,影响着MDSC的增殖、分化及功能.     

Induction of CD4+ and CD8+ anti‐tumor effector T cell responses by bacteria mediated tumor therapy

Facultative anaerobic bacteria like E. coli can colonize solid tumors often resulting in tumor growth retardation or even clearance. Little mechanistic knowledge is available for this phenomenon which is however crucial for optimization and further implementation in the clinic. Here, we show that intravenous injections with E. coli TOP10 can induce clearance of CT26 tumors in BALB/c mice. Importantly, re‐challenging mice which had cleared tumors showed that clearance was due to a specific immune reaction. Accordingly, lymphopenic mice never showed tumor clearance after infection. Depletion experiments revealed that during induction phase, CD8⁺ T cells are the sole effectors responsible for tumor clearance while in the memory phase CD8⁺ and CD4⁺ T cells were involved. This was confirmed by adoptive transfer. CD4⁺ and CD8⁺ T cells could reject newly set tumors while CD8⁺ T cells could even reject established tumors. Detailed analysis of adoptively transferred CD4⁺ T cells during tumor challenge revealed expression of granzyme B, FasL, TNF‐α and IFN‐γ in such T cells that might be involved in the anti‐tumor activity. Our findings should pave the way for further optimization steps of this promising therapy.     

Agonist anti-GITR antibody enhances vaccine-induced CD8(+) T-cell responses and tumor immunity

Immunization of mice with plasmids encoding xenogeneic orthologues of tumor differentiation antigens can break immune ignorance and tolerance to self and induce protective tumor immunity. We sought to improve on this strategy by combining xenogeneic DNA vaccination with an agonist anti-glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) monoclonal antibody (mAb), DTA-1, which has been shown previously both to costimulate activated effector CD4(+) and CD8(+) T cells and to inhibit the suppressive activity of CD4(+)CD25(+) regulatory T cells. We found that ligation of GITR with DTA-1 just before the second, but not the first, of 3 weekly DNA immunizations enhanced primary CD8(+) T-cell responses against the melanoma differentiation antigens gp100 and tyrosinase-related protein 2/dopachrome tautomerase and increased protection from a lethal challenge with B16 melanoma. This improved tumor immunity was associated with a modest increase in focal autoimmunity, manifested as autoimmune hypopigmentation. DTA-1 administration on this schedule also led to prolonged persistence of the antigen-specific CD8(+) T cells as well as to an enhanced recall CD8(+) T-cell response to a booster vaccination given 4 weeks after the primary immunization series. Giving the anti-GITR mAb both during primary immunization and at the time of booster vaccination increased the recall response even further. Finally, this effect on vaccine-induced CD8(+) T-cell responses was partially independent of CD4(+) T cells (both helper and regulatory), consistent with a direct costimulatory effect on the effector CD8(+) cells themselves.     

Celecoxib promotes the efficacy of STING-targeted therapy by increasing antitumor CD8+ T-cell functions via modulating glucose metabolism of CD11b+Ly6G+ cells

Recent studies have shown that activation of the cGAS-STING pathway is a key process in antitumor immune responses and various kinds of STING agonists have been developed for cancer immunotherapy. Despite promising preclinical studies, preliminary clinical results have shown only a modest effect of STING agonists. There is therefore a need to develop more effective treatment strategies. Based on previous observations that COX-2 is frequently overexpressed not only in a variety of cancers but also in tumor myeloid cells and that it suppresses antitumor immunity and promotes tumor survival by producing PGE2, we investigated the antitumor effects of combination therapy with a STING agonist cGAMP and the selective COX-2 inhibitor celecoxib in mouse models. Combination treatment with cGAMP and celecoxib inhibited tumor growth compared with either monotherapy, and the combination therapy induced both local and systemic antitumor immunity. cGAMP treatment decreased PD-1 expression on tumor-infiltrating T-cells and enhanced T-cell activation in tumor-draining lymph nodes regardless of the presence of celecoxib. Meanwhile, although celecoxib treatment did not alter the frequency of CD4⁺CD25⁺Foxp3⁺ regulatory T-cells, it enhanced the expression of costimulatory molecules and glycolysis-associated genes in tumor-infiltrating CD11b⁺Ly6G⁺ cells. Moreover, we also found that celecoxib decreased lactate efflux and increased the frequency of IFN-γ- and TNF-α-producing CD8⁺ T-cells in the tumor microenvironment. Taken together, our findings suggest that combined treatment with celecoxib may be an effective strategy to improve the antitumor efficacy of STING agonists.     

Superior antitumor in vitro responses of allogeneic matched sibling compared with autologous patient CD8+ T cells

Allogeneic cell therapy as a means to break immunotolerance to solid tumors is increasingly used for cancer treatment. To investigate cellular alloimmune responses in a human tumor model, primary cultures were established from renal cell carcinoma (RCC) tissues of 56 patients. In three patients with stable RCC line and human leukocyte antigen (HLA)-identical sibling donor available, allogeneic and autologous RCC reactivities were compared using mixed lymphocyte/tumor cell cultures (MLTC). Responding lymphocytes were exclusively CD8(+) T cells, whereas CD4(+) T cells or natural killer cells were never observed. Sibling MLTC populations showed higher proliferative and cytolytic antitumor responses compared with their autologous counterparts. The allo-MLTC responders originated from the CD8(+) CD62L(high)(+) peripheral blood subpopulation containing naive precursor and central memory T cells. Limiting dilution cloning failed to establish CTL clones from autologous MLTCs or tumor-infiltrating lymphocytes. In contrast, a broad panel of RCC-reactive CTL clones was expanded from each allogeneic MLTC. These sibling CTL clones either recognized exclusively the original RCC tumor line or cross-reacted with nonmalignant kidney cells of patient origin. A minority of CTL clones also recognized patient-derived hematopoietic cells or other allogeneic tumor targets. The MHC-restricting alleles for RCC-reactive sibling CTL clones included HLA-A2, HLA-A3, HLA-A11, HLA-A24, and HLA-B7. In one sibling donor-RCC pair, strongly proliferative CD3(+)CD16(+)CD57(+) CTL clones with non-HLA-restricted antitumor reactivity were established. Our results show superior tumor-reactive CD8 responses of matched allogeneic compared with autologous T cells. These data encourage the generation of antitumor T-cell products from HLA-identical siblings and their potential use in adoptive immunotherapy of metastatic RCC patients.     

Modeling the CD8+ T effector to memory transition in adoptive T-cell antitumor immunotherapy

Adoptive T-cell therapy with CD8(+) CTLs is often characterized by poor persistence of the transferred T cells and limited effector responses. Improved persistence and therapeutic efficacy have been noted when antigen-activated CD8(+) T cells express properties of memory cells. The current study was undertaken to more precisely characterize the development of memory-like CD8(+) T cells from short-term CTLs in vitro and upon transfer in vivo, including their antitumor activity. Ovalbumin (OVA)-specific OT-I CTLs acquired phenotypic and functional properties of memory cells 2 to 3 days later either by lowering the concentration of antigen to a level that does not support primary responses and providing a survival signal through transgenic Bcl-2 in vitro or simply by transferring early day 3 CTLs to antigen-free lymphoid-replete mice. In lymphoid-replete mice, established OVA-expressing E.G7 tumor was rejected by short-term CTLs that simultaneously acquired memory-like properties in secondary lymphoid tissues, where tumor antigen level remained low. Collectively, these data indicate that CTLs readily converted to memory-like cells upon lowering antigen to a concentration that selectively supports memory responses and suggest that such conversion predicts successful adoptive immunotherapy.     

Sustained antigen-specific antitumor recall response mediated by gene-modified CD4+ T helper-1 and CD8+ T cells

Given that specific subsets of T helper 1 (Th1) and T helper 2 (Th2) CD4(+) T cells have been shown to play key roles in tumor rejection models, we wanted to assess the contribution of either Th1 or Th2 CD4(+) cell subtypes for redirected T-cell immunotherapy. In this study, we have developed a novel method involving retroviral transduction and in vitro T-cell polarization to generate gene-engineered mouse CD4(+) Th1 and Th2 cells or T helper intermediate (Thi) cells expressing an anti-erbB2-CD28-zeta chimeric receptor. Gene-modified Th1 and Th2 polarized CD4(+) cells were characterized by the preferential secretion of IFN-gamma and interleukin-4, respectively, whereas Thi cells secreted both cytokines following receptor ligation. In adoptive transfer studies using an erbB2(+) lung metastasis model, complete survival of mice was observed when transduced Th1, Th2, or Thi CD4(+) cells were transferred in combination with an equivalent number of transduced CD8(+) T cells. Tumor rejection was consistently associated with transduced T cells at the tumor site and interleukin-2 secretion. However, the surviving mice treated with gene-modified Th1 CD4(+) cells were significantly more resistant to a subsequent challenge with a different erbB2(+) tumor (4T1.2) implanted s.c. This result correlated with both increased expansion of Th1 CD4(+) and CD8(+) T cells in the blood and a greater number of these cells localizing to the tumor site following rechallenge. These data support the use of gene-modified CD4(+) Th1 and CD8(+) T cells for mediating a sustained antitumor response.