Remembrance of immunology past: conversations with Herman Eisen.

Herman Eisen and Sondra Schlesinger spent several days together in September 2007 in Woods Hole, Massachusetts, talking about immunology, focusing on his remembrances of the field over the more than 60 years of his involvement. This article is an abridged version of those discussions (the full version is available on the Annual Reviews website). It is both an oral history and a written memory of some important but selected areas of immunology.

Manipulating the selection forces during affinity maturation to generate cross-reactive HIV antibodies.

Generation of potent antibodies by a mutation-selection process called affinity maturation is a key component of effective immune responses. Antibodies that protect against highly mutable pathogens must neutralize diverse strains. Developing effective immunization strategies to drive their evolution requires understanding how affinity maturation happens in an environment where variants of the same antigen are present. We present an in silico model of affinity maturation driven by antigen variants which reveals that induction of cross-reactive antibodies often occurs with low probability because conflicting selection forces, imposed by different antigen variants, can frustrate affinity maturation. We describe how variables such as temporal pattern of antigen administration influence the outcome of this frustrated evolutionary process. Our calculations predict, and experiments in mice with variant gp120 constructs of the HIV envelope protein confirm, that sequential immunization with antigen variants is preferred over a cocktail for induction of cross-reactive antibodies focused on the shared CD4 binding site epitope.

Persistent Antigen and Prolonged AKT-mTORC1 Activation Underlie Memory CD8 T Cell Impairment in the Absence of CD4 T Cells.

Recall responses by memory CD8 T cells are impaired in the absence of CD4 T cells. Although several mechanisms have been proposed, the molecular basis is still largely unknown. Using a local influenza virus infection in the respiratory tract and the lung of CD4(-/-) mice, we show that memory CD8 T cell impairment is limited to the lungs and the lung-draining lymph nodes, where viral Ags are unusually persistent and abundant in these mice. Persistent Ag exposure results in prolonged activation of the AKT-mTORC1 pathway in Ag-specific CD8 T cells, favoring their development into effector memory T cells at the expense of central memory T cells, and inhibition of mTORC1 by rapamycin largely corrects the impairment by promoting central memory T cell development. The findings suggest that the prolonged AKT-mTORC1 activation driven by persistent Ag is a critical mechanism underlying the impaired memory CD8 T cell development and responses in the absence of CD4 T cells.

Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation.

During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV) disfavours homotypic bivalent antibody binding. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV.

Promiscuous binding of extracellular peptides to cell surface class I MHC protein.

Algorithms derived from measurements of short-peptide (8-10 mers) binding to class I MHC proteins suggest that the binding groove of a class I MHC protein, such as K(b), can bind well over 1 million different peptides with significant affinity (<500 nM), a level of ligand-binding promiscuity approaching the level of heat shock protein binding of unfolded proteins. MHC proteins can, nevertheless, discriminate between similar peptides and bind many of them with high (nanomolar) affinity. Some insights into this high-promiscuity/high-affinity behavior and its impact on immunodominant peptides in T-cell responses to some infections and vaccination are suggested by results obtained here from testing a model developed to predict the number of cell surface peptide-MHC complexes that form on cells exposed to extracellular (exogenous) peptides.

Interaction of streptavidin-based peptide-MHC oligomers (tetramers) with cell-surface TCRs.

The binding of oligomeric peptide-MHC (pMHC) complexes to cell surface TCR can be considered to approximate TCR-pMHC interactions at cell-cell interfaces. In this study, we analyzed the equilibrium binding of streptavidin-based pMHC oligomers (tetramers) and their dissociation kinetics from CD8(pos) T cells from 2C-TCR transgenic mice and from T cell hybridomas that expressed the 2C TCR or a high-affinity mutant (m33) of this TCR. Our results show that the tetramers did not come close to saturating cell-surface TCR (binding only 10-30% of cell-surface receptors), as is generally assumed in deriving affinity values (K(D)), in part because of dissociative losses from tetramer-stained cells. Guided by a kinetic model, the oligomer dissociation rate and equilibrium constants were seen to depend not only on monovalent association and dissociation rates (k(off) and k(on)), but also on a multivalent association rate (µ) and TCR cell-surface density. Our results suggest that dissociation rates could account for the recently described surprisingly high frequency of tetramer-negative, functionally competent T cells in some T cell responses.

Evolving concepts of specificity in immune reactions.

Our goal is to provide a perspective on current understanding of the origins of specificity in immune reactions, a topic that has intrigued scientists for over a century. A fundamental property of adaptive immune responses is the ability to discriminate among an immense variety of substances by means of antibodies (Abs) and Ab-like receptors on T lymphocytes [T-cell receptors (TCRs)], each able to bind a particular chemical structure [the antigen (Ag)] and not, or only weakly, similar alternatives. Evidence has long existed, however, and has grown, especially recently, that while exhibiting remarkable specificity, many individual Abs and TCRs can also bind a variety of very different ligands. How can Ag recognition by these receptors exercise the great specificity for which they are renowned and yet react with a variety of different ligands (degeneracy)? We critically consider the mechanistic bases for this specificity/degeneracy enigma and also compare and contrast Ag recognition by Abs and TCRs.

Antigen-bearing dendritic cells regulate the diverse pattern of memory CD8 T-cell development in different tissues.

Memory T cells of the effector type (T(EM)) account for the characteristic rapidity of memory T-cell responses, whereas memory T cells of the central type (T(CM)) account for long-lasting, vigorously proliferating memory T-cell responses. How antigen-stimulated (primed) T cells develop into different memory T-cell subsets with diverse tissue distributions is largely unknown. Here we show that after respiratory tract infection of mice with influenza virus, viral antigen associated with dendritic cells (DCs) was abundant in lung-draining lymph nodes (DLN) and the spleen for more than a week but was scant and transient in nondraining lymph nodes (NDLN). Correspondingly, activated CD8 T cells proliferated extensively in DLN and the spleen but minimally in NDLN. Strikingly, however, although most persisting CD8 T cells in DLN and spleen exhibited the T(EM) phenotype, those persisting in NDLN exhibited the T(CM) phenotype. Reducing antigen exposure by depleting DCs at the peak of primary T-cell responses enhanced the development of T(CM), whereas subjecting primed CD8 T cells from NDLN to additional antigen stimulation inhibited T(CM) development. These findings demonstrate that differences in persistence of antigen-bearing DCs in various tissues regulate the tissue-specific pattern of memory CD8 T-cell development. The findings have significant implications for design of vaccines and immunization strategies.

The impact of TCR-binding properties and antigen presentation format on T cell responsiveness.

TCR interactions with cognate peptide-MHC (pepMHC) ligands are generally low affinity. This feature, together with the requirement for CD8/CD4 participation, has made it difficult to dissect relationships between TCR-binding parameters and T cell activation. Interpretations are further complicated when comparing different pepMHC, because these can vary greatly in stability. To examine the relationships between TCR-binding properties and T cell responses, in this study we characterized the interactions and activities mediated by a panel of TCRs that differed widely in their binding to the same pepMHC. Monovalent binding of soluble TCR was characterized by surface plasmon resonance, and T cell hybridomas that expressed these TCR, with or without CD8 coexpression, were tested for their binding of monomeric and oligomeric forms of the pepMHC and for subsequent responses (IL-2 release). The binding threshold for eliciting this response in the absence of CD8 (K(D) = 600 nM) exhibited a relatively sharp cutoff between full activity and no activity, consistent with a switchlike response to pepMHC on APCs. However, when the pepMHC was immobilized (plate bound), T cells with the lowest affinity TCRs (e.g., K(D) = 30 microM) responded, even in the absence of CD8, indicating that these TCR are signaling competent. Surprisingly, even cells that expressed high-affinity (K(D) = 16 nM) TCRs along with CD8 were unresponsive to oligomers in solution. The findings suggest that to drive downstream T cell responses, pepMHC must be presented in a form that supports formation of appropriate supramolecular clusters.

Rapid tolerization of virus-activated tumor-specific CD8+ T cells in prostate tumors of TRAMP mice.

To study T cell responses to tumors in an autochthonous model, we expressed a CD8 T cell epitope SIYRYYGL (SIY) in the prostate of transgenic adenocarcinoma (TRAMP) mice (referred to as TRP-SIY), which spontaneously develop prostate cancer. Naïve SIY-specific CD8 T cells adoptively transferred into TRP-SIY mice became tolerized in the prostate draining lymph nodes. Vaccination of TRP-SIY mice intranasally with influenza virus that expresses the SIY epitope resulted in generation of SIY-specific effector T cells in the lung-draining lymph nodes. These effector T cells expressed TNFalpha and IFNgamma, eliminated SIY peptide-loaded target cells in vivo, and infiltrated prostate tumors, where they rapidly lost the ability to produce effector cytokines. A population of these T cells persisted in prostate tumors but not in lymphoid organs and could be induced to re-express effector functions following cytokine treatment in vitro. These findings suggest that T cells of a given clone can be activated and tolerized simultaneously in different microenvironments of the same host and that effector T cells are rapidly tolerized in the tumors. Our model provides a system to study T cell-tumor interactions in detail and to test the efficacy of cancer immunotherapeutic strategies.

Affinity Inequality among Serum Antibodies That Originate in Lymphoid Germinal Centers.

Upon natural infection with pathogens or vaccination, antibodies are produced by a process called affinity maturation. As affinity maturation ensues, average affinity values between an antibody and ligand increase with time. Purified antibodies isolated from serum are invariably heterogeneous with respect to their affinity for the ligands they bind, whether macromolecular antigens or haptens (low molecular weight approximations of epitopes on antigens). However, less is known about how the extent of this heterogeneity evolves with time during affinity maturation. To shed light on this issue, we have taken advantage of previously published data from Eisen and Siskind (1964). Using the ratio of the strongest to the weakest binding subsets as a metric of heterogeneity (or affinity inequality), we analyzed antibodies isolated from individual serum samples. The ratios were initially as high as 50-fold, and decreased over a few weeks after a single injection of small antigen doses to around unity. This decrease in the effective heterogeneity of antibody affinities with time is consistent with Darwinian evolution in the strong selection limit. By contrast, neither the average affinity nor the heterogeneity evolves much with time for high doses of antigen, as competition between clones of the same affinity is minimal.

A model T-cell receptor system for studying memory T-cell development.

When T-cell clones were first grown in long-term cell culture, each clone was considered to be capable of displaying a limited range of functional activities, constrained by the clones' coreceptor, CD4 or CD8, and the specificity of its antigen-specific receptor (TCR) for one or a few peptides in association with a class I or class II MHC molecule. Subsequent studies, especially with transgenic mice, have shown, however, that T cells expressing the same receptor can be obtained in a variety of differentiated states, including naïve cells, activated effector cells, memory cells, and anergic or tolerant cells, as well as cells with or without a coreceptor. In each of these states T cells can display distinctly different responses to the peptide-MHC (pepMHC) complexes the TCR recognizes. Recently, memory T cells have received considerable attention, in part because of the likelihood that they confer long-term protective immunity against diverse infectious agents and possibly against some forms of cancer. Here we review some recent studies that our colleagues and we have carried out on memory CD8(+) T cells. These studies have made extensive use of cells that express the TCR called 2C. The diverse set of cells expressing the 2C TCR, in mice and in cultured clones and cell lines, are referred to as the 2C system. Before reviewing the studies of memory cells, we summarize the 2C system's main features, including evidence that a large and diverse array of pepMHC complexes, involving at least four class I MHC proteins, can stimulate TCR-mediated responses of 2C cells.

Naïve to memory T-cell differentiation during homeostasis-driven proliferation.

During homeostasis-driven T-cell proliferation, naïve T cells stably acquire the cell surface markers and functional properties of antigen-induced memory T cells. Thus, in T-cell-deficient individuals, homeostasis-driven T-cell proliferation appears to restore only the memory T-cell compartment, whereas the reconstitution of the naïve T-cell compartment depends on de novo T-cell development in the thymus.

Use of siRNAs to prevent and treat influenza virus infection.

Influenza virus causes one of the most prevalent infections in humans. In a typical year, 10-20% of the population in the United States are infected by influenza virus, resulting in up to 40,000 deaths. Current vaccines can prevent illness in approximately 70-80% of healthy individuals under age 65, but the protection rate is much lower in those most susceptible to infection, namely infants, the elderly, and immunocompromised individuals. Although four antiviral drugs have been approved in the United States for treatment and/or prophylaxis of influenza, their use is limited because of concerns about side effects, compliance, and the possible emergence of resistant virus. We found that short interfering RNAs (siRNAs) specific for conserved regions of the influenza virus genome are potent inhibitors of influenza virus replication in both cell lines and embryonated chicken eggs. In this review, we discuss the potential value of siRNAs for preventing and treating influenza virus infections in humans and the challenges that have to be overcome to realize their potential.

Affinity enhancement of antibodies: how low-affinity antibodies produced early in immune responses are followed by high-affinity antibodies later and in memory B-cell responses.

The antibodies produced initially in response to most antigens are high molecular weight (MW) immunoglobulins (IgM) with low affinity for the antigen, while the antibodies produced later are lower MW classes (e.g., IgG and IgA) with, on average, orders of magnitude higher affinity for that antigen. These changes, often termed affinity maturation, take place largely in small B-cell clusters (germinal center; GC) in lymphoid tissues in which proliferating antigen-stimulated B cells express the highly mutagenic cytidine deaminase that mediates immunoglobulin class-switching and sequence diversification of the immunoglobulin variable domains of antigen-binding receptors on B cells (BCR). Of the large library of BCR-mutated B cells thus rapidly generated, a small minority with affinity-enhancing mutations are selected to survive and differentiate into long-lived antibody-secreting plasma cells and memory B cells. BCRs are also endocytic receptors; they internalize and cleave BCR-bound antigen, yielding peptide-MHC complexes that are recognized by follicular helper T cells. Imperfect correlation between BCR affinity for antigen and cognate T-cell engagement may account for the increasing affinity heterogeneity that accompanies the increasing average affinity of antibodies. Conservation of mechanisms underlying mutation and selection of high-affinity antibodies over the ≈200 million years of evolution separating bird and mammal lineages points to the crucial role of antibody affinity enhancement in adaptive immunity.

Koch Institute Symposium on Cancer Immunology and Immunotherapy.

The 12th annual summer symposium of The Koch Institute for Integrative Cancer Research at MIT was held in Cambridge, MA, on June 14th, 1023. The symposium entitled "Cancer Immunology and Immunotherapy" focused on recent advances in preclinical research in basic immunology and biomedical engineering, and their clinical application in cancer therapies. The day-long gathering also provided a forum for discussion and potential collaborations between engineers and clinical investigators. The major topics presented include: (i) enhancement of adoptive cell therapy by engineering to improve the ability and functionality of T-cells against tumor cells; (ii) current therapies using protein and antibody therapeutics to modulate endogenous anti-tumor immunity; and (iii) new technologies to identify molecular targets and assess therapeutic efficacy, and devices to control and target drug delivery more effectively and efficiently.

Loss of IL-7R and IL-15R expression is associated with disappearance of memory T cells in respiratory tract following influenza infection.

Following influenza virus infection, memory CD8 T cells are found in both lymphoid and nonlymphoid organs, where they exhibit striking differences in survival. We have assessed persistence, phenotype, and function of memory CD8 T cells expressing the same TCR in the airways, lung parenchyma, and spleen following influenza virus infection in mice. In contrast to memory CD8 T cells in the spleen, those residing in the airways gradually lost expression of IL-7R and IL-15R, did not respond to IL-7 and/or IL-15, and exhibited poor survival both in vivo and in vitro. Following adoptive transfer into the airways, splenic memory CD8 T cells also down-regulated IL-7R and IL-15R expression and failed to undergo homeostatic proliferation. Thus, although cytokines IL-7 and IL-15 play an essential role in memory CD8 T cell homeostasis in lymphoid organs, the levels of IL-7R and IL-15R expression likely set a threshold for the homeostatic regulation of memory CD8 T cells in the airways. These findings provide a molecular explanation for the gradual loss of airway memory CD8 T cells and heterosubtypic immunity following influenza infection.

Stage-dependent reactivity of thymocytes to self-peptide--MHC complexes.

In mice that express a transgene for the 2C T cell antigen-receptor (TCR) and lack a recombinase-activating gene (2C(+)RAG(-/-) mice) most of the peripheral T cells are CD8(+), a few are CD4(+), and a significant fraction are CD4(-)CD8(-) [double negative (DN)]. The DN 2C cells, like DN T cells that are abundant in various other alphabeta TCR-transgenic mice, appear to be derived directly from DN thymocytes that prematurely express the TCR transgene. The DN 2C cells are virtually absent in mice deficient in major histocompatibility complex class II (MHC-II) but more abundant in mice deficient in MHC-I, suggesting that the DN 2C thymocytes are positively selected by self-peptide-MHC-II (pMHC-II) complexes and negatively selected by self-pMHC-I complexes. The pMHC-I complexes, however, positively select CD8(+) 2C T cells in the same mice. The different effects of thymic pMHC-I on DN and CD8(+) thymocytes are consistent with the finding that DN 2C thymocytes are more sensitive than more mature CD4(+)CD8(+) [double positive (DP)] thymocytes to a weak pMHC-I agonist for the 2C TCR. Together with previous evidence that DP thymocytes respond more sensitively than T cells in the periphery to weak pMHC agonists, the findings suggest progressive decreases in responsiveness to self-pMHC-I complexes as thymocytes develop from DN to DP thymocytes and then to mature naïve T cells in the periphery.

Development of CD4+ T cells expressing a nominally MHC class I-restricted T cell receptor by two different mechanisms.

Differences in T cell receptor (TCR) signaling initiated by interactions among TCRs, coreceptors, and self-peptide-MHC complexes determine the outcome of CD4 versus CD8 lineage of T cell differentiation. The H-2Ld and Kbm3 alloreactive 2C TCR is positively selected by MHC class I Kb and a yet-to-be identified nonclassical class I molecule to differentiate into CD8+ T cells. Here we describe two mechanisms by which CD4+ 2C T cells can be generated in 2C TCR-transgenic mice. In the RAG-/- background, development of CD4+ 2C T cells requires the expression of both I-Ab and the TAP genes, indicating that both MHC class I and II molecules are required for positive selection of these T cells. Notably, only some of the 2C+ RAG-/- mice (approximately 30%) develop CD4+ 2C T cells, with frequencies in individual mice varying from 0.5% to as high as approximately 50%. In the RAG+ background, where endogenous TCRalpha genes are rearranged and expressed, CD4+ 2C T cells are generated because these cells express the 2C TCR as well as additional TCRs, consisting of the 2C TCRbeta and endogenous TCRalpha chains. Similarly, T cells expressing the OT-1 TCR, which is nominally MHC class I-restricted, can also develop into CD4+ T cells through the same two mechanisms. Thus, expression of two TCRs by a single thymocyte, TCR recognition of multiple MHC molecules, and heterogeneity of TCR, coreceptors, and peptide-MHC interactions in the thymus all contribute to the outcome of CD4 versus CD8 lineage development.