Robert J. North

Down-regulation of the antitumor immune response.

Publisher Summary This chapter discusses the immune response to chemically induced, transplantable tumors in syngeneic mice. It deals with those tumors that are immunogenic by virtue of their possession of tumor-specific, transplantation rejection antigens. A framework of evidence has been presented in the chapter that supports the hypothesis of down-regulation of antitumor immune response. Direct evidence for the hypothesis consists of the finding that infusion of suppressor spleen cells from donors with a large tumor can prevent recipient mice from generating a concomitant immune response. Additional direct evidence is being supplied by an ongoing study that shows that complete or partial regression of the Meth A fibrosarcoma that results from appropriately timed exposure to sublethal, whole-body γ-radiation is associated with a prolonged generation of effector T cells and an absence of suppressor T cells. It is suggested that down-regulation of antitumor immunity by suppressor T cells can explain the escape of only of those tumors that are immunogenic enough to evoke the generation of enough effectors T cells to cause tumor regression in the absence of suppressor T cells. This implies that the immunity generated to some tumors is too little too late to cause regression, even in the absence of the negative regulatory influence of suppressor cells. Therefore, successful immunotherapy of some established tumors, besides depending on the employment of agents capable of eliminating suppressor T cells, will also depend on the employment of agents capable of directly augmenting the generation of effectors T cells.

Resistance to fever induction and impaired acute-phase response in interleukin-1β-deficient mice

We used gene targeting in embryonic stem cells to introduce an IL-1 beta null allele in mice. The IL-1 beta-deficient mice develop normally and are apparently healthy and fertile. The IL-1 beta null mice responded normally in models of contact and delayed-type hypersensitivity or following bacterial endotoxin LPS-induced inflammation. The IL-1 beta-deficient mice showed equivalent resistance to Listeria monocytogenes compared with wild-type controls. In contrast, when challenged with turpentine, which causes localized inflammation and tissue injury, the IL-1 beta mutant mice exhibited an impaired acute-phase inflammatory response and were completely resistant to fever development and anorexia. These results highlight a central role for IL-1 beta as a pyrogen and a mediator of the acute-phase response in a subset of inflammatory disease models, and support the notion that blocking the action of a single key cytokine can alter the course of specific immune and inflammatory responses.

Expression of Th1-mediated immunity in mouse lungs induces a Mycobacterium tuberculosis transcription pattern characteristic of nonreplicating persistence

The lung is the primary target of infection with Mycobacterium tuberculosis. It is well established that, in mouse lung, expression of adaptive, Th1-mediated host immunity inhibits further multiplication of M. tuberculosis. Here, real-time RT-PCR was used to define the pattern of expression against time of lung infection of key genes involved in Th1-mediated immunity and of selected genes of M. tuberculosis. Inhibition of bacterial multiplication was preceded by increased mRNA synthesis for IFN-γ and inducible NO synthase (NOS2) and by NOS2 protein synthesis in infected macrophages. Concurrently, the pattern of transcription of bacterial genes underwent dramatic changes. mRNA synthesis increased for α-crystallin (acr), rv2626c, and rv2623 and decreased for superoxide dismutase C (sodC), sodA, and fibronectin-binding protein B (fbpB). This pattern of M. tuberculosis transcription is characteristic of the nonreplicating persistence [Wayne, L. G. & Sohaskey, C. D. (2001) Annu. Rev. Microbiol. 55, 139–163] associated with adaptation of tubercle bacilli to hypoxia in vitro. Based on this similarity, we infer that host immunity induces bacterial growth arrest. In IFN-γ gene-deleted mice, bacterial growth was not controlled; NOS2 protein was not detected in macrophages; sodC, sodA, and fbpB transcription showed no decrease; and acr, rv2626c, and rv2623 transcription increased only at the terminal stages of lung pathology. These findings define the transcription signature of M. tuberculosis as it transitions from growth to persistence in the mouse lung. The bacterial transcription changes measured at onset of Th1-mediated immunity are likely induced, directly or indirectly, by nitric oxide generated by infected macrophages.

Changes in energy metabolism of Mycobacterium tuberculosis in mouse lung and under in vitro conditions affecting aerobic respiration

Transcription profiling of genes encoding components of the respiratory chain and the ATP synthesizing apparatus of Mycobacterium tuberculosis was conducted in vivo in the infected mouse lung, and in vitro in bacterial cultures subjected to gradual oxygen depletion and to nitric oxide treatment. Transcript levels changed dramatically as infection progressed from bacterial exponential multiplication (acute infection) to cessation of bacterial growth (chronic infection) in response to host immunity. The proton-pumping type-I NADH dehydrogenase and the aa3-type cytochrome c oxidase were strongly down-regulated. Concurrently, the less energy-efficient cytochrome bd oxidase was transiently up-regulated. The nitrate transporter NarK2 was also up-regulated, indicative of increased nitrate respiration. The reduced efficiency of the respiratory chain was accompanied by decreased expression of ATP synthesis genes. Thus, adaptation of M. tuberculosis to host immunity involves three successive respiratory states leading to decreased energy production. Decreased bacterial counts in mice infected with a cydC mutant (defective in the cytochrome bd oxidase-associated transporter) at the transition to chronic infection provided initial evidence that the bd oxidase pathway is required for M. tuberculosis adaptation to host immunity. In vitro, NO treatment and hypoxia caused a switch from transcription of type I to type II NADH dehydrogenase. Moreover, cytochrome bd oxidase expression increased, but cytochrome c oxidase expression decreased slightly (nitric oxide) or not at all (hypoxia). These specific differences in respiratory metabolism during M. tuberculosis growth arrest in vitro and in vivo will guide manipulation of in vitro conditions to model bacterial adaptation to host immunity.

Importance of thymus-derived lymphocytes in cell-mediated immunity to infection☆

Abstract Mice made T cell deficient by thymectomy and irradiation, and protected with bone marrow cells (THXB mice) have a greatly reduced capacity to develop antituberculous immunity. In contrast, their capacity to develop anti- Listeria immunity is not impaired. Infection-induced lymphoid cell proliferation in the spleen is greatly reduced in THXB mice infected with either organism. The reduced capacity of THXB mice to develop antituberculous immunity and to show increased lymphoid cell proliferation in response to a tuberculous infection can be restored by an intraperitoneal injection of normal syngeneic thymocytes. In contrast, reduced lymphoid cell proliferation in THXB mice infected with Listeria cannot be restored with thymocytes. Nevertheless, the spleen cells from Listeria -infected donors with the capacity to transfer anti- Listeria immunity to normal recipients are destroyed in vitro by anti-θ serum and complement, thus showing they are T cells. The apparent contradiction is explained in terms of a residual T cell population in THXB mice which is large enough to generate anti- Listeria , but not antituberculous immunity.

The Murine Antitumor Immune Response and Its Therapeutic Manipulation

Publisher Summary This chapter presents a framework of results showing that a host bearing a progressive immunogenic tumor responds immunologically to its tumor by generating a state of concomitant antitumor immunity. Because a host with an already established tumor represent the real situation that a therapist is confronted with, the possible possession by the host of a state of concomitant immunity would need to be considered in any attempt to cause regression of the tumor be immunotherapy. The results presented in this chapter leave little doubt that a syngeneic host can generate an immune response against its immunogenic tumor, but that the response is “down-regulated” by suppressor T cells before enough effector T cells are generated to destroy the tumor. This conclusion is based on the results of two main lines of investigation that involved the use of an adoptive immunization model illustrated diagrammatically. The chapter argues that an analysis of the generation and decay of concomitant immunity is essential for an understanding of why immunogenic tumors grow progressively in their immunocompetent hosts and why these tumors fail to regress in response to active or passive immunotherapy.

Properties and protective value of the secondary versus primary T helper type 1 response to airborne Mycobacterium tuberculosis infection in mice

Mice immunized against Mycobacterium tuberculosis (Mtb) infection by curing them of a primary lung infection were compared with naive mice in terms of the ability to generate a Th1 cell immune response and to control growth of an airborne Mtb challenge infection. Immunized mice generated and expressed Th1 cell immunity several days sooner than naive mice, as demonstrated by an earlier increase in the synthesis in the lungs of mRNA for Th1 cytokines and for inducible nitric oxide synthase, an indicator of macrophage activation. This Th1 cytokine/mRNA synthesis was accompanied by an earlier accumulation of Mtb-specific Th1 cells in the lungs and the presence of CD4 T cells in lesions. An earlier generation of immunity was associated with an earlier inhibition of Mtb growth when infection was at a 1-log lower level. However, inhibition of Mtb growth in immunized, as well as in naive, mice was not followed by resolution of the infection, but by stabilization of the infection at a stationary level. The results indicate that there is no reason to believe that the secondary response to an Mtb infection is quantitatively or qualitatively superior to the primary response.

Carbon flux rerouting during Mycobacterium tuberculosis growth arrest

A hallmark of the Mycobacterium tuberculosis life cycle is the pathogens ability to switch between replicative and non‐replicative states in response to host immunity. Transcriptional profiling by qPCR of ∼ 50 M. tuberculosis genes involved in central and lipid metabolism revealed a re‐routing of carbon flow associated with bacterial growth arrest during mouse lung infection. Carbon rerouting was marked by a switch from metabolic pathways generating energy and biosynthetic precursors in growing bacilli to pathways for storage compound synthesis during growth arrest. Results of flux balance analysis using an in silico metabolic network were consistent with the transcript abundance data obtained in vivo. Similar transcriptional changes were seen in vitro when M. tuberculosis cultures were treated with bacteriostatic stressors under different nutritional conditions. Thus, altered expression of key metabolic genes reflects growth rate changes rather than changes in substrate availability. A model describing carbon flux rerouting was formulated that (i) provides a coherent interpretation of the adaptation of M. tuberculosis metabolism to immunity‐induced stress and (ii) identifies features common to mycobacterial dormancy and stress responses of other organisms.

Gamma-irradiation facilitates the expression of adoptive immunity against established tumors by eliminating suppressor T cells.

SummaryIt was found that sublethal (550 rad) whole-body γ-irradiation of mice bearing established immunogenic tumors enabled tumor-sensitized spleen cells infused intravenously 1 h later to cause complete tumor regression in all mice. In contrast, γ-irradiation alone caused only a temporary halt in tumor growth, and immune cells gave practically no therapeutic effect at all. This result was obtained with the SA1 sarcoma, Meth A fibrosarcoma, P815 mastocytoma, and P388 lymphoma. Additional experiments with the Meth A fibrosarcoma revealed that the spleen cells from tumor-immune donors that caused tumor regression in γ-irradiated recipients were T cells, as evidenced by their functional elimination by treatment with anti-Thy-1.2 antibody and complement. It was shown next that adoptive T-cell-mediated regression of tumors in γ-irradiated recipients was inhibited by an intravenous infusion of spleen cells from donors with established tumors, but not by spleen cells from normal donors. The spleen cells that suppressed the expression of adoptive immunity were functionally eliminated by treatment with anti-Thy-1.2 antibody and complement. Moreover, they were destroyed by exposing the tumor-bearing donors to 500 rad of γ-radiation 24 h before harvesting their spleen cells. The results are consistent with the interpretation that γ-radiation facilitates the expression of adoptive T-cell-mediated immunity against established tumors by eliminating a population of tumor-induced suppressor T cells from the tumor-bearing recipient.

Expression levels of Mycobacterium tuberculosis antigen‐encoding genes versus production levels of antigen‐specific T cells during stationary level lung infection in mice

Mycobacterium tuberculosis lung infection in mice was controlled at an approximately stationary level after 20 days of log linear growth. Onset of stationary level infection was associated with the generation by the host of T helper type 1 (Th1) immunity, as evidenced by the accumulation of CD4 Th1 cells specific for the early secretory antigen (ESAT‐6) of M. tuberculsosis encoded by esat6, and for a mycolyl transferase (Ag85B) encoded by fbpB. CD4 T cells specific for these antigens were maintained at relatively high numbers throughout the course of infection. The number of CD4 T cells generated against ESAT‐6 was larger than the number generated against Ag85B, and this was associated with a higher transcription level of esat6. The total number of transcripts of esat6 increased during the first 15 days of infection, after which it decreased and then approximately stabilized at 106·5 per lung. The total number of fbpB transcripts increased for 20 days of infection before decreasing and then approximately stabilizing at 104·8 per lung. The number of transcripts of esat6 per colony‐forming unit of M. tuberculosis fell from 8·6 to 0·8 after day 15, and of fbpB from 0·3 to less than 0·02 after day 10, suggesting that at any given time during stationary level infection the latter gene was expressed by a very small percentage of bacilli. Expressed at an even lower level was an M. tuberculosis replication gene involved in septum formation (ftsZ), indicating that there was no significant turnover of the M. tuberculosis population during stationary level infection.