On antigen-specific signals, immune class regulation and energetics: Report III from the workshops on foundational concepts of immune regulation.

This is a report from a one-week workshop held in Athens, Greece in July of 2022. The workshop aimed to identify emerging concepts relevant to the fundamentals of immune regulation and areas for future research. Theories of immune regulation emphasize the role of T cell help or co-stimulation (signal 2). The workshop participants considered how new data on the characteristics of agonist antigens, the role of the antigen receptor signals (signal 1) in driving fate decisions, the effect of energetics on immunity and a better understanding of class-control in the immune response, may impact theories of immune regulation. These ideas were discussed in the context of tumour immunology, autoimmunity, pregnancy and transplantation. Here we present the discussions as a narrative of different viewpoints to allow the reader to join the conversation. These discussions highlight the evolving understanding of the nature of specific antigen recognition and how both antigen-specific and non-specific mechanisms impact immune responses.

The Problem of Host and Pathogen Genetic Variability for Developing Strategies of Universally Efficacious Vaccination against and Personalised Immunotherapy of Tuberculosis: Potential Solutions?

Rational vaccination against and immunotherapy of any infectious disease requires knowledge of how protective and non-protective immune responses differ, and how immune responses are regulated, so their nature can be controlled. Strong Th1 responses are likely protective against M tuberculosis. Understanding how immune class regulation is achieved is pertinent to both vaccination and treatment. I argue that variables of infection, other than PAMPs, primarily determine the class of immunity generated. The alternative, non-PAMP framework I favour, allows me to propose strategies to achieve efficacious vaccination, transcending host and pathogen genetic variability, to prevent tuberculosis, and personalised protocols to treat disease.

The historical postulate: Is it the basis, at the level of the system, for self-nonself discrimination?

Burnet envisaged the early presence of self-antigens in development, or 'the history' of an animal, ablates the animal's ability to immunologically respond against them. Lederberg added the idea that the continuous presence of self-antigens is required to maintain tolerance throughout life. We refer to Lederberg's proposal as 'The Historical Postulate'. The mechanism of central tolerance, as now understood, is consistent with The Historical Postulate. Some observations, reflecting peripheral tolerance, appear inconsistent with this postulate. For example, some foreign peripheral tissues, grafted onto an animal before the immune system develops, can be rejected as the immune system matures. The original two-signal model of lymphocyte activation was proposed in part because it accounted for peripheral tolerance in a manner consistent with The Historical Postulate. We proposed that lymphocyte activation required antigen-mediated lymphocyte cooperation, whereas antigen would inactivate lymphocytes when insufficient in number to achieve activation. We argue here that the exceptions to The Historical Postulate can be explained by the two-signal model of lymphocyte activation: they reflect the existence of greater numbers of lymphocytes specific for these antigens than for natural peripheral antigens, and so are outside the physiological limits important in selecting through evolution this mechanism of peripheral tolerance. We argue that a consideration of whether The Historical Postulate is valid is important, even if only valid within certain understandable limits. The currently popular DAMP model of CD4 T cell activation is, strictly speaking and in a manner we discuss, in violation of this postulate.

Can interruption/withdrawl of anti-retroviral therapy provide personalized immunotherapy against HIV-1?

We propose a treatment of HIV-1+ individuals designed to harness protective immunity, lead to viral containment, and so render the individual minimally infectious. A few HIV-infected individuals, 'elite controllers', generate a stable Th1, cytotoxic T lymphocyte response that contains the virus. Most infected individuals, in the absence of therapy, first generate a similarly protective response that evolves with time a Th2 component, associated with antibody production and loss of viral control. Cessation of anti-retroviral treatment after three years results in viral rebound in most, but about one in seven individuals contains the virus, so-called post-treatment controllers. We suggest an understanding, of how the Th1/Th2 phenotype of immune responses is controlled, can explain these different outcomes and leads us to propose a non-invasive, personalized strategy of immunotherapy. We propose that monitoring the relative prevalence of HIV-1 specific IgG1 and IgG2 antibodies can provide a biomarker for deciding when to interrupt/withdraw anti-retroviral therapy to optimally harness protective immunity.

On T cell development, T cell signals, T cell specificity and sensitivity, and the autoimmunity facilitated by lymphopenia.

We propose a framework to explain how T cells achieve specificity and sensitivity, how the affinity of the TcR peptide/MHC interaction controls positive and negative thymic selection and mature T cell survival, and whether antigen-dependent activation and inactivation takes place. Two distinct types of signalling can lead to mature T cell multiplication. One requires the TcR to recognize with a certain affinity an antigen-derived peptide, an agonist peptide, bound to an MHC molecule. The other, the tonic signal, leads to naïve T cell survival and modest proliferation if the T cell successfully competes for endogenous, self-peptide/MHC ligands, involving lower affinity TCR/ligand interactions. Many suggest lymphopenia contributes to autoimmunity by increasing the strength of TcR-tonic signalling, and so activation of anti-self T cells. We suggest T cell activation requires antigen-mediated cooperation between T cells. Increased tonic signalling under lymphopenic conditions facilitates T cell proliferation and so antigen-dependent cooperation and activation of anti-self T cells.

Does T Cell Activation Require a Quorum of Lymphocytes?

Recent reports suggest a quorum of T cells is required to activate T lymphocytes and that this requirement may help explain why scarce lymphocytes, specific for peripheral self-antigen, are rarely activated by Ag. This proposal runs counter to the commonly held framework that the Ag-dependent, but CD4 T lymphocyte-independent, activation of CD8 T lymphocytes, and the activation of CD4 T lymphocytes themselves, can occur when a single CD8 or CD4 T lymphocyte encounters Ag under appropriately dangerous circumstances. We argue that a review of older literature often ignored, as well as of contemporary studies, supports the quorum concept and is difficult to reconcile with the Danger Model.

The history of the two-signal model of lymphocyte activation: A personal perspective.

The first ideas leading to The Two-Signal Model of lymphocyte activation were published 50 years ago, but the model was not realized in one sitting. I describe the three phases that led to its contemporary formulations. A motivation underlying all these models was to generate a minimal description of what is required for antigen to inactivate and activate mature lymphocytes that, at the same time, accounts for how peripheral tolerance is achieved. I suggest the two signal model has not only provided a substantiated framework for understanding how antigen interacts differently with B cells and CD8 T cells, to result in their inactivation and activation, but its postulates are pertinent to contemporary issues concerning the inactivation and activation of CD4 T cells.

On how the immune system preferentially interacts with antigen-specific molecules bound to antigen over unbound molecules, with emphasis on B cell receptor signalling.

Antigen-specific molecules of the immune system, namely antibodies, the membrane immunoglobulins (mIgs) of B cells and T cell receptors (TcRs), can all signal their interaction with antigen. There are different mechanisms by which this signalling could occur. These mechanisms can be divided into two general categories: allosteric and non-allosteric. In allosteric mechanisms, the monovalent binding of the antigen to the receptor triggers a conformational change at the binding site that is propagated to an invariant part of the receptor, a change recognized by a sensing unit. We argue allosteric mechanisms are implausible. Non-allosteric mechanisms depend on steric effects due to the antigen's size and/or multivalency. We consider two non-allosteric mechanisms by which the mIg of B cells has been envisaged to signal its interaction with antigen: the popular cross-linking model and the dissociation activation model. We argue, on the basis of both experimental observations and physiological considerations, that the dissociation activation model, developed by Reth and his colleagues, is uniquely plausible.

Is the Framework of Cohn's 'Tritope Model' for How T Cell Receptors Recognize Peptide/Self-MHC Complexes and Allo-MHC Plausible?

Cohn has developed the tritope model to describe how distinct domains of the T cell receptor (TcR) recognize peptide/self-MHC complexes and allo-MHC. He has over the years employed this model as a framework for considering how the TcR might mediate various signals [1-5]. In a recent publication [5], Cohn employs the Tritope Model to propose a detailed mechanism for the T cell receptor's involvement in positive thymic selection [5]. During a review of this proposal, I became uneasy over the plausibility of the underlying framework of the Tritope Model. I outline here the evolutionary considerations making me question this framework. I also suggest that the proposed framework underlying the Tritope Model makes strong predictions whose validity can most probably be assessed by considering observations reported in the literature.

The number of responding CD4 T cells and the dose of antigen conjointly determine the TH1/TH2 phenotype by modulating B7/CD28 interactions.

Our previous in vivo studies show that both the amount of Ag and the number of available naive CD4 T cells affect the Th1/Th2 phenotype of the effector CD4 T cells generated. We examined how the number of OVA-specific CD4 TCR transgenic T cells affects the Th1/Th2 phenotype of anti-SRBC CD4 T cells generated in vivo upon immunization with different amounts of OVA-SRBC. Our observations show that a greater number of Ag-dependent CD4 T cell interactions are required to generate Th2 than Th1 cells. We established an in vitro system that recapitulates our main in vivo findings to more readily analyze the underlying mechanism. The in vitro generation of Th2 cells depends, as in vivo, upon both the number of responding CD4 T cells and the amount of Ag. We demonstrate, using agonostic/antagonistic Abs to various costimulatory molecules or their receptors, that the greater number of CD4 T cell interactions, required to generate Th2 over Th1 cells, does not involve CD40, OX40, or ICOS costimulation, but does involve B7/CD28 interactions. A comparison of the level of expression of B7 molecules by APC and CD4 T cells, under different conditions resulting in the substantial generation of Th1 and Th2 cells, leads us to propose that the critical CD28/B7 interactions, required to generate Th2 cells, may directly occur between CD4 T cells engaged with the same B cell acting as an APC.

A Plausible Framework Reveals Potential Similarities in the Regulation of Immunity against Some Cancers and Some Infectious Agents: Implications for Prevention and Treatment.

Different frameworks, which are currently employed to understand how immune responses are regulated, can account for different observations reported in the classical literature. I have argued that the predominant frameworks, employed over the last two/three decades to analyze the circumstances that determine whether an immune response is generated or this potential is ablated, and that determine the class of immunity an antigen induces, are inconsistent with diverse classical observations. These observations are "paradoxical" within the context of these frameworks and, consequently, tend to be ignored by most contemporary researchers. One such observation is that low and high doses of diverse types of antigen result, respectively, in cell-mediated and IgG antibody responses. I suggest these paradoxes render these frameworks implausible. An alternative framework, The Threshold Hypothesis, accounts for the paradoxical observations. Some frameworks are judged more plausible when found to be valuable in understanding findings in fields beyond their original compass. I explore here how the Threshold Hypothesis, initially based on studies with chemically well-defined and "simple antigens", most often a purified protein, can nevertheless shed light on diverse classical and more recent observations in the fields of immunity against cancer and against infectious agents, thus revealing common, immune mechanisms. Most cancers and some pathogens are best contained by cell-mediated immunity. The success of the Threshold Hypothesis has encouraged me to employ it as a basis for proposing strategies to prevent and to treat cancer and those infectious diseases caused by pathogens best contained by a cell-mediated attack.

Direct demonstration of CD4 T cell cooperation in the primary in vivo generation of CD4 effector T cells.

Many observations bear upon the cellular and molecular requirements for CD4 T cell activation. The interaction of CD4 T cells with dendritic cells (DC), central to the induction of most immune responses, is the most studied. However, leukocytes other than DC can dramatically affect the induction and differentiation of CD4 T cells into effector cells. We recently provided indirect evidence that in vivo CD4 T cooperation facilitates the activation of CD4 T cells. Here, we demonstrate that the activation of CD4 T cells, specific for the hen egg lysozyme (HEL)(105) (-120) peptide, is optimally achieved when BALB/c mice are immunized with additional MHC class II-binding HEL peptides in incomplete Freund's adjuvant. This cooperation cannot be mimicked by the coadministration of LPS or of an agonistic antibody to CD40, at the time of immunization. In contrast, OX40-OX40L interactions are necessary for CD4 T cell cooperation in that an OX40 agonistic antibody can replace, and an OX40L-blocking antibody can abrogate, CD4 T cell cooperation in situations where such cooperation would otherwise enhance the activation of CD4 T cells.

The activation, by antigen, of naïve TCR transgenic CD4 T cells cultured at physiological, rather than artificially high, frequencies more accurately reflects the in vivo activation of normal numbers of naïve CD4(+) T cells.

The majority of in vitro studies investigating the activation of naïve TCR transgenic T cells routinely employ an artificially high frequency of such cells. To assess whether employing high frequencies of TCR transgenic cells in vitro accurately reflects the in vivo activation of a normal number of T cells, we cultured between 300 and 3×10(6) Rag2(-/-) DO11.10 T cells per well under otherwise identical conditions. We find that those T cells cultured at low frequencies proliferate more and are more potently activated, as assessed by the expression of CD44 and CD62L, each giving rise to a much larger number of cytokine producing cells, comparable to the number generated in vivo when a normal number of CD4(+) T cells are activated. The effect of T cell frequency on the level of their activation was not due to differences in MHCII or CD80/86 expression by B cells, the major APC population present, nor to increased death of B cells in high frequency cultures. Taken together, our observations illustrate the necessity of culturing naïve TCR transgenic CD4(+) T cells at a physiological frequency if one is to more accurately recapitulate the in vivo activation of naïve CD4(+) T cells.

Facing the Increased Prevalence of Antibiotic-Resistant M. tuberculosis: Exploring the Feasibility of Realising Koch's Aspiration of Immunotherapy of Tuberculosis.

Koch attempted to treat tuberculosis in the late 1800s by administering an antigenic extract derived from the pathogen to patients. He hoped to bolster the patient's protective immunity. The treatment had diverse results. In some, it improved the patient's condition and in others led to a worsening state and even to death. Koch stopped giving his experimental treatment. I consider here three issues pertinent to realizing Koch's vision. Rational immunotherapy requires a knowledge of what constitutes protective immunity; secondly, how on-going immune responses are regulated, so the patient's immunity can be modulated to become optimally protective; thirdly, a simple methodology by which treatment might be realized. I deliberately cast my account in simple terms to transcend barriers due to specialization. The proposed immunotherapeutic treatment, if realizable, would significantly contribute to overcoming problems of treatment posed by antibiotic resistance of the pathogen.

Analyzing some concepts of immune regulation of the last three decades: Fostering greater research resilience despite the information overload. A personal view.

There is considerable interest in whether increased investment in science, made by society, pays dividends. Some plausibly argue the increased rate of production of information results in an ossification of the canon. Reports, challenging the canon, fall by the wayside. The field thus becomes increasingly complex, reflecting not so much the reality of nature but how we investigate the subject. I suggest that focusing on and resolving the paradoxes evident within a canon will free the logjam, resulting in more resilient research. Immunology is among the fastest growing of biological sciences and is, I suggest, an appropriate case study. I examine the commonly accepted frameworks employed over the last three decades to address two major, related immunological questions: what determines whether antigen activates or inactivates CD4 T cells, and so whether immune responses are initiated or this potential ablated; secondly, what determines the Th subset to which the activated Th cells belong, thus determining the class of immunity generated. I show there are major paradoxes within these frameworks, neglected for decades. I propose how research focused on resolving paradoxes can be better fostered, and so support the evolution of the canon. This perspective is pertinent in facing critical issues on how immune responses are regulated, and to more general issues of both the philosophy of science and of science policy.The last section is in response to questions and comments of the reviewers. It brings together several considerations to express my view: the same frameworks, formulated in response to the two questions, are useful in understanding the regulation of the immune response against model antigens, against self and foreign antigens, those of tumors and of pathogens.

CD4 T cell cooperation is required for the in vivo activation of CD4 T cells.

We address here the role of CD4 T cell cooperation in the activation of CD4 T cells. Administration of aggregated hen egg lysozyme (HEL) without microbial adjuvant to BALB/c mice normally generates cytokine-producing CD4 T cells specific for the HEL major peptide, HEL(105-120), as well as CD4 T cells specific for HEL non-major peptides. The prior administration of HEL(105-120) ablates the generation of cytokine-secreting CD4 T cells specific for HEL(105-120), as well as the CD4 T cells specific for HEL non-major peptides, normally generated upon HEL challenge. Thus, the activation of HEL non-major peptide-specific CD4 T cells appears to depend upon the HEL(105-120)-specific CD4 T cell population. In contrast, when HEL(105-120) and saline-treated mice are challenged with HEL coupled to ovalbumin (OVA), CD4 T cell responses to HEL non-major peptides and to OVA are the same, whereas treated mice still do not generate cytokine-secreting cells specific for HEL(105-120). We infer that the administration of HEL(105-120) does not generate regulatory cells capable of down-regulating CD4 T cell responses to HEL and OVA peptides. OVA-specific CD4 T cells restore the generation of HEL non-major peptide-specific T cells in the absence of HEL major peptide-specific T cells. We conclude that the generation of CD4 T cells producing IL-2, IFN-gamma and IL-4 requires CD4 T cell cooperation and that this cooperation is not mediated simply by CD40-CD40L interactions. We also conclude from these observations that there is no requirement for a microbial or danger signal for CD4 T cell activation.

What information is needed to design effective vaccination against intracellular pathogens causing chronic disease?

An appreciation of global features of immune regulation may lead to vaccination strategies effective in a genetically diverse population against a number of intracellular pathogens that cause chronic disease. Such global strategies appear more straightforward than strategies requiring a detailed knowledge of the specificity of 'protective T-cells'. Global strategies may be effective against the virus, bacteria and protozoa that respectively cause AIDS, tuberculosis and the leishmaniases.

Antigen presenting B cells facilitate CD4 T cell cooperation resulting in enhanced generation of effector and memory CD4 T cells.

We show that the in vivo generation of cytokine-producing CD4 T cells specific for a given major histocompatibility class-II (MHCII)-binding peptide of hen egg lysozyme (HEL) is facilitated when mice are immunized with splenic antigen presenting cells (APC) pulsed with this HEL peptide and another peptide that binds a different MHCII molecule. This enhanced generation of peptide-specific effector CD4 T cells requires that the same splenic APC be pulsed with both peptides. Pulsed B cells, but not pulsed dendritic cells (DCs), can mediate CD4 T cell cooperation, which can be blocked by disrupting OX40-OX40L (CD134-CD252) interactions. In addition, the generation of HEL peptide-specific CD4 T cell memory is greater when mice are primed with B cells pulsed with the two peptides than with B cells pulsed with the HEL- peptide alone. Based on our findings, we suggest CD4 T cell cooperation is important for vaccine design, underlies the phenomenon of "epitope-spreading" seen in autoimmunity, and that the efficacy of B cell-depletion in the treatment of human cell-mediated autoimmune disease is due to the abrogation of the interactions between autoimmune CD4 T cells that facilitates their activation.

Immunization of newborn and adult mice with low numbers of BCG leads to Th1 responses, Th1 imprints and enhanced protection upon BCG challenge.

Neonatal bacille Calmette-Guerin (BCG) vaccination is widely employed to protect against tuberculosis. Predominant Th1 but not mixed Th1/Th2 responses are thought to be protective. If so, effective vaccination must cause Th1 imprints. The immune system of infants differs from that of adults and such differences could critically affect neonatal vaccination. We demonstrate that BCG infection of infant and adult mice produces similar responses. Infection with low and high numbers of BCG, respectively, leads to sustained Th1 and mixed Th1/Th2 responses. Low-dose but not high-dose infection also results in Th1 imprints, guaranteeing a Th1 response upon high-dose challenge, and resulting in optimal bacterial clearance. Our observations on low-dose Th1 imprinting are intriguing in the context of the well-known madras trial. In this trial, the highest dose of BCG, which had insignificant side effects, was administered to over 250,000 human subjects. This high-dose vaccination resulted in insignificant protection against tuberculosis.

Different immune correlates associated with tumor progression and regression: implications for prevention and treatment of cancer.

Observations show that humans and animals respond immunologically to most cancers. Why does the immune system then fail to control cancer? We argue from the literature that there is a commonality in the regulation of responses against most murine tumors, and that a major mechanism of escape may be deviation of an effective Th1, cytotoxic T lymphocyte response to a less effective response with a Th2 component. We examined this hypothesis with two well-studied murine tumors. We found, following primary tumor implantation, that resistance correlates with Th1 responses and IgG2a antibody production and progression with mixed Th1/Th2 responses and production of IgG1 and IgG2a antibodies. Resistance is associated with a modulation of the anti-tumor response towards the Th1 pole in both systems. We conclude that the immune responses against these two tumors are in accord with our hypothesis, and argue that this is likely to be true of many human and murine tumors. The correlation of IgG isotype of anti-tumor antibody with the Th1/Th2 nature of the anti-tumor response readily allows one to longitudinally monitor the changing nature of the anti-tumor response. We suggest that such monitoring can guide immunotherapy to maximize the effectiveness of the host's immune response against cancer.