In recent years the combined use of chemotherapy and immunotherapy, collectively termed chemoimmunotherapy, has emerged as a promising treatment option for patients with cancer. Antibiotics are commonly used to reduce infection-related complications in patients undergoing chemotherapy. Intriguingly, accumulating evidence has implicated gut microbiota as a critical determinant of host antitumor immune responses, raising the question as to whether the use of broad-spectrum antibiotics would invariably diminish tumor response to chemoimmunotherapies. We investigated the impact of antibiotics on the therapeutic outcomes of cyclophosphamide (CTX) chemotherapy and adoptive T-cell therapy (ACT) where CTX was used as the host-conditioning regimen in mice. We show that antibiotic prophylaxis dampened the endogenous T cell responses elicited by CTX, and reduced the efficacy of CTX against B-cell lymphoma. In the ACT setting, antibiotics administration impaired the therapeutic effects of adoptively transferred tumor-specific CD4+ T cells in mice with implanted colorectal tumors. In contrast, long-term antibiotic exposure did not affect the efficacy of ACT using CD19-targeting chimeric antigen receptor (CAR) T cells in mice with systemic B-cell lymphoma, although it correlated with prolonged CAR expression and sustained B-cell aplasia. Our study demonstrates that chemoimmunotherapies may have variable reliance on intestinal microbiota for T cell activation and function, and thus have different sensitivities to antibiotic prophylaxis. These findings may have implications for the judicial use of antibiotics in cancer patients receiving chemoimmunotherapies.

SUMMARY The inhibitory effects of cancer on T cell metabolism have been well established, but the metabolic impact of immunotherapy on tumor cells is poorly understood. Here, we developed a CD4+ T cell-based adoptive immunotherapy protocol that was curative for mice with implanted colorectal tumors. By conducting metabolic profiling on tumors, we show that adoptive immunotherapy profoundly altered tumor metabolism, resulting in glutathione depletion and accumulation of reactive oxygen species (ROS) in tumor cells. We further demonstrate that T cell-derived tumor necrosis factor alpha (TNF-α) can synergize with chemotherapy to intensify oxidative stress and tumor cell death in an NADPH (nicotinamide adenine dinucleotide phosphate hydrogen) oxidase-dependent manner. Reduction of oxidative stress, by preventing TNF-α-signaling in tumor cells or scavenging ROS, antagonized the therapeutic effects of adoptive immunotherapy. Conversely, provision of pro-oxidants after chemotherapy can partially recapitulate the antitumor effects of T cell transfer. These findings imply that reinforcing tumor oxidative stress represents an important mechanism underlying the efficacy of adoptive immunotherapy.

The beta-domain of metallothionein-3 (MT3) has been reported to be crucial to the neuron growth inhibitory bioactivity. Little detailed three-dimensional structural information is available to present a reliable basis for elucidation on structure-property-function relationships of this unique protein by experimental techniques. So, molecular dynamics simulation is adopted to study the structure of beta-domain of MT3. In this article, a 3D structural model of beta-domain of MT3 was generated. The molecular simulations provide detailed protein structural information of MT3. As compared with MT2, we found a characteristic conformation formed in the fragment (residue 1-13) at the N-terminus of MT3 owing to the constraint induced by 5TCPCP9, in which Pro7 and Pro9 residues are on the same side of the protein, both facing outward and the two 5-member rings of prolines are arranged almost in parallel, while Thr5 is on the opposite side. Thr5 in MT3 is also found to make the first four residues relatively far from the fragment (residue 23-26) as compared with MT2. The simulated structure of beta-domain of MT3 is looser than that of MT2. The higher energy of MT3 than that of MT2 calculated supports these conclusions. Simulation on the four isomer arising from the cis- or trans-configuration of 6CPCP9 show that the trans-/trans-isomer is energetic favorable. The partially unfolding structure of beta-domain of MT3 is also simulated and the results show the influence of 6CPCP9 sequence on the correct folding of this domain. The correlations between the bioactivity of MT3 and the simulated structure as well as the folding of beta-domain of MT3 are discussed based on our simulation and previous results.

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Increased availability of homeostatic cytokines is considered a major mechanism by which lymphodepletion enhances the efficacy of adoptive T cell therapy (ACT). IL-7 is one such cytokine capable of augmenting the function of tumor-reactive CD8+ T cells. However, whether host-derived IL-7 plays a role in driving the proper function of CD4+ T cells in an ACT setting remains unclear. Here we report that lymphodepleting chemotherapy by cyclophosphamide (CTX) does not lead to increased availability of the endogenous IL-7 in mice. Despite of a paucity of IL-7 in the immune milieu, CTX preconditioning allowed adoptively transferred naïve tumor-specific CD4+ T cells to undergo effector differentiation and regain IL-7Rα expression, giving rise to IL-7-responsive polyfunctional CD4+ effector cells. Correspondingly, supplementation of exogenous recombinant IL-7 markedly amplified and sustained polyfunctional CD4+ effector cells, resulting in improved therapeutic outcome in a mouse lymphoma model. We further demonstrated that the immune-enhancing effects of IL-7 were also applicable to donor CD4+ T cells pre-activated under Th1 polarizing condition. These findings suggest caution in relying on the endogenous IL-7 to enhance donor T cell expansion and persistence after lymphodepleting chemotherapy, and highlight the usefulness of recombinant IL-7 as an adjuvant for adoptive immunotherapy.

DatabaseThe structures of the a-domain in Cd 7 -hGIF and the solo Cd 4 -a-domain have been deposited in the Protein Data Bank under the numbers 2FJ4 and 2FJ5, respectively *These authors contributed equally to this work (Received 16 October 2008, revised 19 March 2009, accepted 24 April 2009) doi:10.1111/j.1742-4658.2009 Human neuronal growth inhibitory factor (hGIF) is able to inhibit the outgrowth of neurons. As compared with the amino acid sequences of metallothionein 1 ⁄ 2, hGIF contains two insertions: a Thr at position 5 and an acidic hexapeptide EAAEAE(55-60) close to the C-terminus. Moreover, all mammalian growth inhibitory factor sequences contain a conserved CPCP(6-9) motif. Previous studies have demonstrated that the TCPCP(5-9) motif is pivotal to its bioactivity, but no specific role has been assigned to the unique EAAEAE(55-60) insert. To investigate the potential structural and biological significance of the EAAEAE(55-60) insert, several mutants were constructed and investigated in detail. Notably, deletion of the acidic insert (the D55-60 mutant) reduced the inhibitory activity, whereas the bioactivities of other mutants did not change much. Then, spectroscopic characterization and molecular dynamics simulation were performed to investigate the potential causes of the reduced bioactivity of the D55-60 mutant. It was found that the domain-domain interaction mechanism of hGIF was different from that of metallothionein 2. It was also shown that the acidic insert could regulate the interdomain interactions in hGIF, leading to the structural change in the b-domain, which resulted in the alteration of the solvent accessibility and metal release ability, thus playing an important role in the biological activity of hGIF. Our studies provided useful information on the domain-domain interaction at the molecular level for the first time, and shed new light on the mechanism of the bioactivity of hGIF.Abbreviations AD, Alzheimer's disease; GIF, neuronal growth inhibitory factor; hGIF, human neuronal growth inhibitory factor; hMT1g, human MT1 isoform g; MT, metallothionein; SNOC, S-nitrosocysteine; ZINCON, 2-(2-hydroxy-5-sulfoformazyl) benzoic acid.

Human metallothionein-3 (hMT3), also named human neuronal growth inhibitory factor (hGIF), is attractive due to its distinct neuronal growth inhibitory activity, which is not shown by other human MT isoforms. It has been reported that the neuronal growth inhibitory activity arises from the N-terminal beta-domain rather than its C-terminal alpha-domain. However, previous bioassay results have shown that the single beta-domain is less effective at inhibiting the neuron growth than that in intact hMT3 on a molar basis, which suggests that the alpha-domain is indispensable to the neuronal growth inhibitory activity of hMT3. In order to confirm this assumption, we constructed two domain-hybrid mutants, the beta(MT3)-beta(MT3) mutant and the beta(MT3)-alpha(MT1) mutant, and investigated their structural and metal binding properties by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, etc. The results showed that stability of the Cd(3)S(9) cluster of the beta(MT3)-beta(MT3) mutant decreased significantly while the Cd(3)S(9) cluster of the beta(MT3)-alpha(MT1) mutant had a similar stability and solvent accessibility to that of hMT3. Interestingly, the bioassay results showed that the neuronal growth inhibitory activity of the beta(MT3)-beta(MT3) mutant decreased significantly, while the beta(MT3)-alpha(MT1) mutant showed similar inhibitory activity to hMT3. Based on these results, we conclude that the alpha-domain is indispensable and plays an important role in modulating the stability of the metal cluster in the beta-domain by domain-domain interactions, thus influencing the bioactivity of hMT3.