A hypothesis for the existence of two types of tuberculosis, reflecting two distinct types of immune failure to control the pathogen, based upon prevalence of mycobacterium-specific IgG subclasses.

Most people infected by Mycobacterium tuberculosis, about 90%, contain the pathogen and are healthy. Most investigators have concluded that pathogen-specific Th1 cells contribute to protection. Pulmonary tuberculosis, the most prevalent form of disease, is associated with destructive granulomas, the formation of which also appears to involve Th1 cells. In what sense then do the two Th1 components of the response, in healthy infected individuals and patients, differ? An insight into this question might provide clues for attaining effective vaccination and better treatment. We approached this question by examining the relative prevalence of different IgG isotypes among anti-mycobacterium-specific antibodies in patients and healthy infected individuals as a surrogate marker for the Th1/Th2 phenotype of the response. Our observations lead us to agree that healthy infected individuals generate a predominant Th1 response. Our observations also lead us to propose that many patients make a similar kind of response as healthy infected individuals, but that this response is too weak to contain the infection. We refer to such individuals as having type I tuberculosis. Other patients appear to have a greater and detrimental Th2 component to their immune response than that of healthy infected individuals. We refer to these individuals as having type II tuberculosis. This proposal that there are two types of tuberculosis, reflecting two distinct types of failure by the immune system, will, if correct, be pertinent to vaccine design, treatment of tuberculosis and in making further progress in our understanding the genetics of susceptibility to M. tuberculosis.

Immune Class Regulation and Its Medical Significance Part II of a Report of a Workshop on Foundational Concepts of Immune Regulation.

We discussed different proposals for how the nature of the Th1/Th2 phenotype of an immune response is determined, and favoured one, the Threshold Hypothesis, as plausible and so useful as the basis for further discussions. The activation of a target CD4 T cell can be facilitated by helper CD4 T cells when the CD4 T cells interact via an antigen-presenting cell. The Threshold Hypothesis states that tentative and robust antigen-mediated CD4 T cell cooperation results in the target CD4 T cell, respectively giving rise, upon activation, to Th1 and Th2 cells. We primarily discussed four topics. We briefly discussed in the background section certain limitations of the Th1/Th2 paradigm in understanding immune class regulation, and the remarkable anti-inflammatory properties of human IgG4 antibody. Secondly, we assessed the role of class II MHC molecules in determining the number of mature CD4 T cells and so affecting the Th1/Th2 phenotype of immune responses. We also discussed the controversial role of CD8 T cells in affecting the Th1/Th2 phenotype of responses to MHC and other antigens, and the potential role of their relative scarcity in neonates in biasing responses towards an antibody, Th2 mode. Lastly, we examined the regulation of the Th1/Th2 phenotype of both primary and ongoing immune responses in the context of the intriguing proposal that antigen initially generates different classes/subclasses of immunity and then selects, by a feedback mechanism, the most effective class. We found this interesting idea difficult to reconcile with various observations.

Immunological Tolerance. Part I of a Report of a Workshop on Foundational Concepts of Immune Regulation.

This report, the first of two, arose from a one-week workshop directed at discussing concepts of immune regulation, and focuses on immunological tolerance. We first outline the major ideas we thought sufficiently plausible to provide a context for discussing more controversial issues around tolerance. We then report on our discussion of different experiments that appear paradoxical in terms of the different, contemporary models of CD4 T cell inactivation/activation, and how such observations might be resolved in terms of insights provided by these contemporary models. These discussions bear on the plausibility of the Pathogen-Associated Molecular Pattern (PAMP), Danger and Two Step, Two Signal Models for the activation of naïve CD4 T cells. Some of the observations considered appear paradoxical in terms of the PAMP and Danger Models, but not with the Two Step, Two Signal Model. For example, genetically immunodeficient mice have been given foreign, sterile ectopic grafts, and the immune system allowed to develop once these grafts were well-healed in, and so in the absence of PAMPs or danger. The grafts were rejected, unexpected on the PAMP or Danger Models. We also discussed considerations and observations bearing on the widely held idea that antigen must crosslink the membrane Ig receptors of a B cell to initiate the generation of signal 1, or the alternative possibility that monovalent binding by antigen can do so. We favored the latter possibility, and discussed a particular model, "the Elbow Model," for how this might be achieved.

A Conversation with Cohn on the Activation of CD4 T Cells.

Despite an agreement on most issues surrounding models for how lymphocytes are activated and inactivated, and arising out of the 1970 Two Signal Model of lymphocyte activation, Cohn and I have different perspectives on two critical issues concerning the activation of CD4 T cells. One issue is the origin of the first effector T helper (eTh) cells, postulated by both of us to be required to optimally activate precursor Th (pTh), that is naïve CD4 T cells, to further generate eTh cells. The second issue arises from our agreement that the antigen-dependent CD4 T cell cooperation, that we both postulate is required to activate naïve CD4 T (pTh) cells, most likely is mediated by the operational recognition of linked epitopes. Although agreeing on the centrality of this operational mechanism, we disagree about how it might be realized at the molecular/cellular level. I respond here to issues raised by Cohn concerning these two mechanistic questions, in his response to my recent article on the activation and inactivation of mature CD4 T cells.

The activation and inactivation of mature CD4 T cells: a case for peripheral self-nonself discrimination.

The establishment of central tolerance to most self-antigens results in a repertoire of mature peripheral lymphocytes specific for foreign and peripheral self-antigens. The framework that single, mature lymphocytes are inactivated by antigen, whereas their activation requires lymphocyte cooperation, accounts for diverse observations and incorporates a mechanism of peripheral tolerance. This framework accounts for the generalizations that the sustained activation by antigen of most B cells and CD8 T cells requires CD4 T helper cells; in the absence of CD4 T cells, antigen can inactivate these B cells and CD8 T cells. In this sense, CD4 T cells are the guardians of the fate of most B cells and CD8 T cells when they encounter antigen. I argue here that the single-lymphocyte/multiple-lymphocyte framework for the inactivation/activation of lymphocytes also applies to CD4 T cells. I consider within this framework a model for the activation/inactivation of CD4 T cells that is consistent with the large majority of contemporary observations, including significant clinical observations. I outline the grounds why I feel this model is more plausible than the contemporary and predominant pathogen-associated molecular pattern (PAMP) and Danger Models for CD4 T cell activation. These models are based upon what I consider the radical premise that self-nonself discrimination does not exist at the level of mature CD4 T cells. I explain why I feel this feature renders the PAMP and Danger Models somewhat implausible. The model I propose, in contrast, is conservative in that it embodies such a process of self-nonself discrimination.

On the mechanism determining the TH1/TH2 phenotype of an immune response, and its pertinence to strategies for the prevention, and treatment, of certain infectious diseases.

It is well recognized that the physiological/pathological consequences of an immune response, against a foreign or a self-antigen, are often critically dependent on the class of immunity generated. Here we focus on how antigen interacts with the cells of the immune system to determine whether antigen predominantly generates Th1 or Th2 cells. We refer to this mechanism as the 'decision criterion' controlling the Th1/Th2 phenotype of the immune response. A plausible decision criterion should account for the variables of immunization known to affect the Th1/Th2 phenotype of the ensuing immune response. Documented variables include the nature of the antigen, in terms of its degree of foreignness, the dose of antigen and the time after immunization at which the Th1/Th2 phenotype of the immune response is assessed. These are quantitative variables made at the level of the system. In addition, the route of immunization is also critical. I describe a quantitative hypothesis as to the nature of the decision criterion, referred to as the Threshold Hypothesis. This hypothesis accounts for the quantitative variables of immunization known to affect the Th1/Th2 phenotype of the immune response generated. I suggest and illustrate how this is not true of competing, contemporary hypotheses. I outline studies testing predictions of the hypothesis and illustrate its potential utility in designing strategies to prevent or treat medical situations where a predominant Th1 response is required to contain an infection, such as those caused by HIV-1 and by Mycobacterium tuberculosis, or to contain cancers.

Anti-IL-4 antibody therapy causes regression of chronic lesions caused by medium-dose Leishmania major infection in BALB/c mice.

Experimental infection of BALB/c mice with a high number of Leishmania major parasites results in a predominant Th2 response and rapidly progressing, non-healing lesions. Disease can be prevented in such mice by simple therapies, such as administering neutralizing anti-IL-4 or anti-CD4 antibody prior to or around the time of infection, but not once the infection is well established. Established infections can be resolved by combined therapies, such as intralesional administration of massive doses of IL-12 and of anti-leishmanial drugs. We explored the possibility of using simple therapies to cure mice with stable, chronic and large lesions, a state that corresponds more closely to human cutaneous leishmaniasis than does the rapidly progressing model. The anti-parasite immune responses of mice bearing such chronic lesions have a mixed Th1/Th2 phenotype. Administration of either anti-IL-4 or anti-CD4 antibody alone results in the reliable regression of such lesions even when large. Cured mice display a dominant Th1 response with increased L. major-specific IgG2a antibody, increased production of IL-12p40 and of nitric oxide by macrophages, indicating increased parasiticidal activity. Cured mice resist a normally pathogenic L. major challenge. These findings may have implications for treatment of human cutaneous leishmaniasis and other chronic infectious diseases caused by intracellular pathogens.

Immune elimination of Leishmania major in mice: implications for immune memory, vaccination, and reactivation disease.

Infection of susceptible BALB/c mice with a large, moderate, or low number of Leishmania major parasites respectively results in progressive disease, the formation of substantial but stable lesions, denoted as borderline disease, and the absence of a visible lesion. Infection with a low number of parasites results over the long term in either subclinical infections or an asymptomatic state. Subclinical mice produce a predominant Th1 response and are resistant to challenge, in contrast to their asymptomatic counterparts. Statistical and other evidence suggest that the asymptomatic state can arise from a subclinical state following parasite clearance, with consequent loss of resistance. Cell transfer studies demonstrate unequivocally that immune cells from subclinical mice can protect naive mice against a pathogenic challenge and can clear the parasite, leaving the mice susceptible to a rechallenge infection. This susceptibility is associated with the disappearance of both parasite-specific effector and memory T cells from secondary lymphoid organs. These findings have implications for vaccination, maintenance of memory, and prevention of reactivation disease.

More antigen-dependent CD4(+) T cell / CD4(+) T cell interactions are required for the primary generation of Th2 than of Th1 cells.

Mechanisms controlling the Th1 / Th2 phenotype of a primary immune response are often discussed assuming that the generation of Th1 and Th2 cells from the common CD4(+) precursor T helper (pTh) involves an interaction of this pTh cell with an antigen-presenting cell (APC) in the form of a two-cell interaction. Other studies suggest that the outcome of this two-cell interaction is modified by the presence of other T cells. No study has analyzed primary immune responses generated in normal, non-TcR transgenic mice, following the administration of a non-infectious antigen administered without adjuvant. We show that the Th1 / Th2 phenotype of such a primary response, generated in lethally irradiated recipients reconstituted with a variety of unprimed spleen cells, depends conjointly on the amount of antigen and number of unprimed syngeneic CD4(+) T cells present, with higher amounts and numbers favoring the generation of Th2 cells. Our observations show how these quantitative variables control in an interdependent manner the Th1 / Th2 phenotype of a primary immune response, and bear upon the mechanism by which this phenotype is determined.

Distinct immunity in patients with visceral leishmaniasis from that in subclinically infected and drug-cured people: implications for the mechanism underlying drug cure.

Significant levels of IgG3 and IgG4 and high levels of IgG1 leishmania-specific antibody differentiated the immune states in 10 patients with visceral leishmaniasis from those of virtually all 20 drug-cured and 18 subclinically infected subjects, whereas the level of IgG2 antibody was nondiscriminating. The most extreme "subclinically infected" outlier subsequently developed disease. Overall, the immune states in subclinically infected and drug-cured persons were mutually indistinguishable but were readily distinguished from those of patients. These findings may have implications for the immunologic mechanism underlying drug cure in visceral leishmaniasis.

A case for a neonatal, low-dose BCG vaccination trial.

The rational design of a successful vaccination strategy against tuberculosis requires certain kinds of information and must take account of several considerations: (i) the nature of the immune response that protects the large majority of individuals infected by Mycobacterium tuberculosis, designated as healthy contacts, must be defined and distinguished from that in tuberculosis patients, whose immune system must have failed; (ii) the vaccination strategy must incorporate a way of priming the immune system to guarantee in all individuals this protective response, normally generated in healthy contacts, upon natural infection by M. tuberculosis; (iii) the strategy must incorporate a mechanism for ensuring that the effectiveness of this priming is not abrogated by exposure to environmental mycobacteria; and (iv) the strategy must take account of the fact that the vaccinated population is genetically heterogeneous, and that individuals will therefore respond variably to most standard vaccination protocols. We describe a tentative proposal for how these interrelated problems might be solved and discuss predictions of this tentative vaccination strategy. Critical testing of the neonatal, low-dose BCG vaccination strategy can only be achieved by a field trial and we outline the considerations underlying this proposal.

Contemporary models for peripheral tolerance and the classical 'historical postulate'.

The formulators of Clonal Selection Theory proposed that what distinguishes self from foreign antigens is their early appearance in development, and their continuous presence thereafter. This early presence of a self antigen in an animals life history was envisaged to ensure that lymphocytes specific for the self antigen were silenced rather than activated. I call this idea 'the historical postulate'. Contemporary models for the activation/inactivation of helper T cells implicitly violate this postulate. I argue against such a violation and for a model of T helper cell activation/inactivation that reconciles contemporary observations with the postulate.

The Th1/Th2 nature of concurrent immune responses to unrelated antigens can be independent.

We tested the independence hypothesis, namely that the Th1/Th2 nature of concurrent immune responses, generated in the same secondary lymphoid organ to non-cross-reacting Ags, can be independently determined. Some infectious agents and some adjuvants contain modulatory molecules that affect the Th1/Th2 nature of immune responses in a non-Ag-specific manner. We therefore excluded infectious agents as Ags and the use of adjuvants to generate immune responses. We first show that the dose of xenogeneic RBC administered i.v. determines the Th1/Th2 nature of the splenic immune response. Low doses generate a virtually exclusive Th1 response, whereas a higher dose induces either a mixed Th1/Th2 or a predominantly Th2 response, and stimulates the production of specific Abs. We immunized individual mice simultaneously with a low dose of one kind of xenogeneic RBC and with a higher dose of another non-cross-reacting xenogeneic RBC and assessed the Th1/Th2 nature of the immune responses generated in the spleen to each kind of RBC. The Th1/Th2 nature of the response to each RBC in doubly immunized mice was indistinguishable from that of the corresponding immune response in singly immunized mice. We discuss the significance of our findings for understanding immune class regulation, and the possible reasons why such independence is not always seen.

A valid ELISPOT assay for enumeration of ex vivo, antigen-specific, IFNgamma-producing T cells.

We describe an ELISPOT technique for the detection of antigen specific IFNgamma-producing T cells. The technique is performed on spleen cells plated directly ex vivo into ELISPOT trays without an in vitro pre-culture step. Thus, the assay is likely to reflect the in vivo activity of the cells. We have found that very high cell densities (at least 10(6) cells/well) are required for optimal detection of spot forming cells, and only at a high density of cells is the number of spots detected linearly related to the number of primed cells plated. If lower numbers of antigen primed cells are used, then unprimed spleen cells from syngeneic mice can be added to the well to increase the cell density. Under these conditions, we find that the number of spots is linearly proportional to the number of primed cells plated, even if these are well below a million cells/well. Experiments with MHC congenic mice indicate that the high density of spleen cells required to obtain optimal spot formation reflects a requirement for an MHC restricted function, probably efficient antigen presentation to T cells. The formation of IFNgamma spots is antigen dependent and abrogated by depleting the antigen primed cells of T cells. We conclude that this linear assay can be used to efficiently detect ex vivo antigen-specific IFNgamma-producing T cells.

A two-step, two-signal model for the primary activation of precursor helper T cells.

I present here a new model for the primary activation of precursor helper T cells. Observations demonstrate that the immune system learns not to respond to extrathymic, organ-specific self-antigens because of their early appearance in development. The immune system thus discriminates between peripheral self-antigens and foreign antigens and, when mature, usually makes an immune response against only the latter. Contemporary models for the activation and inactivation of T helper (Th) function do not account for such discrimination. The model proposed here is consistent with contemporary findings and incorporates a mechanism of peripheral self-nonself discrimination.

Parasite dose determines the Th1/Th2 nature of the response to Leishmania major independently of infection route and strain of host or parasite.

Leishmania major causes cutaneous leishmaniasis in mice and man. Infection of mice with relatively low or high numbers of parasites leads respectively to parasite containment, associated with a Th1, cell-mediated response, or progressive disease, associated with a Th2, antibody response in all circumstances studied. These include different parasite strains, different routes of infection, and different hosts previously classified as susceptible, resistant or of intermediate susceptibility. This dose dependency appears to reflect a general rule. We argue that this rule may allow the design of a vaccination strategy that is effective among a genetically diverse population, and that it imposes severe constraints upon proposals for the nature of the "decision criterion" determining whether antigen induces a Th1 or Th2 response.

Mycobacterial dose defines the Th1/Th2 nature of the immune response independently of whether immunization is administered by the intravenous, subcutaneous, or intradermal route.

It is believed that cell-mediated immunity alone can contain Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. The induction of antibody, or of a mixed cell-mediated/humoral response, is associated with tuberculous disease. It is therefore important to determine the conditions of immunization with bacille Calmette Guérin (BCG), the attenuated strain of Mycobacterium bovis used to vaccinate humans against tuberculosis, that optimally induces an exclusive cell-mediated, Th1 response. Such a determination will then allow an assessment of whether the generation of such an exclusive Th1 response results in the generation of a Th1 imprint against mycobacteria. This Th1 imprint would ensure that the Th1 response is predominant following any challenge. We therefore tested the proposition that the dose of mycobacteria used for immunization generally determines the Th1/Th2 nature of the ensuing response. Our results demonstrate that relatively low doses lead to an almost exclusive cell-mediated, Th1 response, while higher doses induce a mixed Th1/Th2 response. Furthermore, the dependence on dose is independent of whether BCG is administered intravenously, subcutaneously, or intradermally. The implications of our findings to understanding how different classes of immunity are induced, to the epidemiology of tuberculosis, and to the design of effective vaccination strategies are discussed.

Distinct immunological states in murine cutaneous leishmaniasis by immunising with different amounts of antigen: the generation of beneficial, potentially harmful, harmful and potentially extremely harmful states.

Infection of BALB/c mice with a standard and substantial number of Leishmania major parasites results in progressive disease, following the induction of a parasite-specific Th2 response. These mice have been designated as "susceptible" on this basis. We show that distinct types of immune response can be generated in "susceptible" BALB/c mice depending upon the number of parasites employed for infection, and that the pathophysiological consequences of such distinct responses are dramatically different. Infection with very low numbers of parasites results in the exclusive induction of a cell-mediated, Th1 response, and the generation of resistance to the standard and substantial challenge. Spleen cells from such resistant mice can confer resistance upon normal mice when transferred to them, but these spleen cells do not contain T cells expressing DTH or Th1 effector cells that produce IFN gamma on short term culture (48 hrs) with parasite antigen. The immune response in this case appears to result in the virtual elimination of parasites from the lymph node draining the site of infection and, by implication, from the infected mouse. We suggest that such elimination results in the absence of antigen stimulation and hence of effector T cells, and that "memory Th1 cells" are responsible for the capacity of spleen cells to confer resistance on normal mice. We predict such mice will not suffer parasitemia upon immune suppression, i.e. are not susceptible to reactivation disease. This is the "beneficial state". In contrast to this infection with a very low number of parasites infection with a low number usually results in one of two states: (i) The generation of a response with a very small Th2 component, production of a small amount of antibody, chronic parasitemia and hence chronic generation of parasite-specific effector Th1/Th2 cells, or (ii) The generation of a response with a greater Th2 component, the production of more antibody, the formation of a frank lesion, and the long term generation of a stable, mixed Th1/Th2 response. We refer to the latter state as borderline leishmaniasis in analogy with borderline leprosy. Parasites can be recovered from the draining lymph node in both these cases many months after infection. We therefore believe that mice infected with a low number of parasites, that harbour a chronic subclinical infection, will suffer reactivation disease upon immune suppression, and we consequently designate the state generated as potentially harmful. We consider mice with borderline disease to be in a harmful state. Mice immunised with high doses of parasite antigen produce in the long term Th2 responses, whereas those immunised with lower doses produce Th1 responses. Mice immunised to produce a Th2 response were subsequently infected with a very low number of parasites that is normally contained. The generation of a Th2 response results in the generation of a Th2 imprint, such that the response to the low dose infection is modulated from a Th1 to a Th2 mode, resulting in progressive disease. We argue that immunisation/vaccination, resulting in a state that deviates the protective response to a non-protective mode, may result in epidemics. Such a state has the potential for being extremely harmful.

Towards a strategy of universally efficacious vaccination against pathogens uniquely susceptible to cell-mediated attack.

Infection by some intracellular parasites is contained only by cell-mediated immunity, and yet antibody is produced at the expense of the cell-mediated response upon natural infection, leading to chronic or fatal disease. Effective vaccination must therefore generate an immunological imprint ensuring a strong and stable cell-mediated response upon infection. Such diseases include leprosy, tuberculosis, the leishmaniasis and AIDS (Kaplan and Cohn (1986) Int. Rev. Exp. Pathol. 28, 45-78; Surcel et al. (1994) Immunology 81, 171-176; Pearson et al. (1983) Rev. Infect. Dis. 5, 907-927; Clerici and Shearer (1993) Immunol. Today 14, 107-111). BALB/C mice are susceptible to Leishmania major, a protozoan that causes cutaneous leishmaniasis in man, by the criterion that substantial infection results in antibody production and progressive disease (Locksley and Scott (1991) Immunoparasitology Today, A58-A61; J.N. Menon and P.A. Bretscher, unpublished data). Infection of BALB/C mice with very few parasites results in an exclusive cell-mediated, Th1-like response and resistance to an ordinarily pathogenic, high dose challenge. This resistance is associated with a strong and stable cell-mediated response (Bretscher et al. (1992) Science 257, 539-542; J.N. Menon and P.A. Bretscher, unpublished data). The generation of this Th1 imprint by low dose infection has been achieved with three very different strains of the parasite. There is a similar dependency of susceptibility and resistance on relative parasite dose in 'susceptible' and 'resistant' mice and in mice of 'intermediate susceptibility'. For example, 'resistant' mice are resistant to substantial infection but succumb to infection with very high doses of parasites. We therefore propose that infection of a genetically diverse population with a very low dose of viable parasites, that does not induce antibody in any individual, will either induce cell-mediated immunity and contain the parasite, or the parasite will grow until it reaches the threshold required to induce cell-mediated immunity, thereby generating the required imprint. Low dose infection may thus constitute universally efficacious vaccination. The pertinence of these observations to improving the efficacy of BCG vaccination against tuberculosis is discussed.

Characterization of the immunological memory state generated in mice susceptible to Leishmania major following exposure to low doses of L. major and resulting in resistance to a normally pathogenic challenge.

BALB/c mice are susceptible to a high-dose infection of the protozoan Leishmania major, which induces a parasite-specific antibody, Th2-like response, exclusive of a significant and protective cell-mediated Th1 component. We have shown, in contrast, that infection with a low number of parasites induces cell-mediated immunity exclusive of antibody production, and results in resistance to substantial subsequent high-dose infection. Low-dose exposure thus constitutes effective vaccination. In the present study, we analyze lymphokine production by parasite-specific T cells from those low-dose exposed, resistant mice and from normal, susceptible mice following high-dose infection. Two findings stand out. First, the parasite-specific T cells in mice rendered resistant appear not to be in an activated, effector state at the time of parasite challenge, as assessed by lack of lymphokine production on short-term stimulation with parasite antigens, but to be rather in a memory state. Second, the ratio of parasite antigen-dependent production of interferon-gamma to that of interleukin-4 by spleen cells of low-dose exposed and normal mice upon high-dose challenge takes a dramatically different course. This ratio is similar in both groups of mice shortly after challenge, but increases dramatically in the resistant and declines dramatically in the control mice over a period of weeks, such that these ratios differ by about 60-fold 12 weeks after the high-dose challenge. In addition, we show that a similar state of resistance occurs following low-dose infection with a more virulent strain of L. major. In toto, our observations suggest that resistance may be generally achievable by low-dose exposure and may be associated with a memory state which, when activated by parasite challenge, results in the evolution of the response over weeks such that the protective, Th1 component becomes ever more dominant over the Th2 component.