Paul V. Lehmann

Induction of TH1 and TH2 Immunity in Neonatal Mice

The neonatal period has been thought of as a window in ontogeny, during which the developing immune system is particularly susceptible to tolerization. In the present study, the classic system for induction of neonatal tolerance to protein antigens was reexamined in mice. The presumably tolerogenic protocol was found to trigger a vigorous T helper cell type 2 (TH2) immune response. Thus, neonatal “tolerization” induces immune deviation, not tolerance in the immunological sense. Neonates are not immune privileged but generate TH2 or TH1 responses, depending on the mode of immunization.

Single-Cytokine-Producing CD4 Memory Cells Predominate in Type 1 and Type 2 Immunity

The patterns of Ag-induced cytokine coexpression in normal, in vivo-primed CD4 memory T cells has remained controversial because the low frequency at which these cells occur has effectively prevented direct ex vivo measurements. We have overcome this limitation by using two-color cytokine enzyme-linked immunospot assays and computer-assisted image analysis. We found CD4 memory cells that simultaneously expressed IL-2, IL-3, IL-4, IL-5, and IFN-γ to be rare (0–10%). This cytokine segregation was seen in adjuvant-induced type 1, type 2, and mixed immunity to OVA, in Leishmania infection regardless of the Ag dose used or how long after immunization the assay was performed. The data suggest that type 1 and type 2 immunity in vivo is not mediated by classic Th1 or Th2 cells but by single-cytokine-producing memory cells.

The human immune system in hu-PBL-SCID mice

Severe combined immunodeficiency (SCID) mice can be stably grafted with human peripheral blood lymphocytes, creating hu-PBL-SCID chimeras; essentially, these are mice with a human immune system. Here, Magdalena Tary-Lehmann, Andrew Saxon and Paul Lehmann discuss the immunobiology of these chimeras. The authors propose that hu-PBL-SCID chimerism evolves in two phases. During the first three weeks after grafting, many of the injected cells survive and the human immune system is functional. Subsequently, anti-mouse-reactive clones are selected and the immune system becomes nonfunctional. The implications of this scenario for the utilization of the hu-PBL-SCID model are discussed.

Direct Visualization of Cytokine-Producing Recall Antigen-Specific CD4 Memory T Cells in Healthy Individuals and HIV Patients

We have used computer-assisted cytokine ELISA spot analysis to measure the frequencies, the type of cytokine, and the amount of cytokine produced by individual recall Ag-specific CD4 memory cells in freshly isolated blood. We studied the memory cells specific for tetanus toxoid and purified protein derivative in 18 healthy individuals and in 22 HIV-infected patients on highly active antiretroviral therapy (HAART). In healthy individuals, the frequency, cytokine signature, and cytokine production per cell of these memory cells were stable over time. Although it is presently unclear whether the maintenance of the memory T cell pool depends upon Ag persistence, cross-reactive Ag stimulation, or cytokine-driven bystander stimulations and expansions, our data strongly argue for a stable memory cell pool in healthy individuals. In HIV patients, however, the frequency of these memory cells was a function of the viral load. The decreased numbers of functional memory cells in patients with high viral loads might provide one mechanism behind the immunodeficient state. Although the cytokine output per cell was unaffected in most patients (20 of 24), in some patients (4 of 24) it was >100-fold reduced, which might provide an additional mechanism to account for the reduced immunocompetence of these patients. The ability to visualize directly and quantify the cytokine produced by the low frequency memory cells in freshly isolated blood that have been physiologically stimulated by Ag should aid comprehensive studies of the Ag-specific memory cell pool in vivo, in health and disease.

Quantification of Self-Recognition in Multiple Sclerosis by Single-Cell Analysis of Cytokine Production

Identifying and quantifying autoaggressive responses in multiple sclerosis (MS) has been difficult in the past due to the low frequency of autoantigen-specific T cells, the high number of putative determinants on the autoantigens, and the different cytokine signatures of the autoreactive T cells. We used single-cell resolution enzyme-linked immunospot (ELISPOT) assays to study, directly ex vivo, proteolipid protein (PLP)-specific memory cell reactivity from MS patients and controls. Overlapping 9-aa-long peptides, spanning the entire PLP molecule in single amino acid steps, were used to determine the frequency and fine specificity of PLP-specific lymphocytes as measured by their IFN-γ and IL-5 production. MS patients (n = 22) responded to 4 times as many PLP peptides as did healthy controls (n = 22). The epitopes recognized in individual patients, up to 22 peptides, were scattered throughout the PLP molecule, showing considerable heterogeneity among MS patients. Frequency measurements showed that the number of PLP peptide-specific IFN-γ-producing cells averaged 11 times higher in MS patients than in controls. PLP peptide-induced IL-5-producing T cells occurred in very low frequencies in both MS patients and controls. This first comprehensive assessment of the anti-PLP-Th1/Th2 response in MS shows a greatly increased Th1 effector cell mass in MS patients. Moreover, the highly IFN-γ-polarized, IL-5-negative cytokine profile of the PLP-reactive T cells suggests that these cells are committed Th1 cells. The essential absence of uncommitted Th0 cells producing both cytokines may explain why therapeutic strategies that aim at the induction of immune deviation show little efficacy in the established disease.

Intratumor CpG-Oligodeoxynucleotide Injection Induces Protective Antitumor T Cell Immunity

Tumor cells are typically poorly immunogenic. The same mechanisms that evolved to avoid the induction of immune responses against self tissues, and, hence, autoimmune disease, also have to be overcome for immune therapy of cancer. Toll-like receptor-activating microbial products such as CpG motif containing DNA are among the primary stimuli that the immune system uses to distinguish between infectious nonself (that is to be attacked) and noninfectious self (that must not be attacked). We tested in a murine RMA lymphoma/C57BL/6 model whether providing the infectious nonself context in a tumor–by injecting CpG-oligodeoxynucleotides directly into the tumor–would elicit a protective antitumor response. Complete remission of established solid tumors was achieved in immune competent mice, but not in T cell/B cell-deficient RAG-1 knockout mice. Intratumor injection of CpG-oligodeoxynucleotides was shown to induce a tumor-specific CD4+ and CD8+ T cell response of the type 1 effector class, and T cells adoptively transferred the protection to RAG-1 knockout mice. The data show that intratumor injection of CpG-oligodeoxynucleotides is a promising strategy for rendering tumors immunogenic.

Protective Anti‐Helicobacter Immunity Is Induced with Aluminum Hydroxide or Complete Freund’s Adjuvant by Systemic Immunization

To determine whether systemic immunization against Helicobacter pylori could be achieved with an adjuvant approved for human use, the efficacy of vaccination with Helicobacter antigen in combination with aluminum hydroxide (AlOH) was evaluated in a murine model of Helicobacter infection. Immunization with antigen and AlOH induced interleukin-5-secreting, antigen-specific T cells, and immunization with antigen and complete Freunds adjuvant induced interferon-gamma-secreting, antigen-specific T cells, as determined by ELISPOT assay. Both immune responses conferred protection after challenge with either H. pylori or H. felis, as confirmed by the complete absence of any bacteria, as assessed by both histology and culture of gastric biopsy samples. Protection was antibody independent, as demonstrated with antibody-deficient muMT mice (immunoglobulin-gene knockout mice), and CD4(+) spleen T cells from immunized mice were sufficient to transfer protective immunity to otherwise immunodeficient rag1(-/-) recipients. These results suggest an alternative and potentially more expeditious strategy for development of a human-use H. pylori vaccine.

A T Cell Clone’s Avidity Is a Function of Its Activation State

At present it is unclear how Ag dose-dependent T cell functions, such as cytokine production, reflect TCR affinity and how the signal strength afforded by the Ag dose affects the kinetics of cytokine production by the individual T cell. We used a computer-assisted ELISPOT approach to address these issues. IFN-γ release by a clonal population of CD4 T cells was monitored on a clonal population of APC while titrating the nominal peptide. The frequency of cytokine-producing cells, the net per-cell output of cytokine, and the onset of cytokine production were each found to be functions of the signal strength. Sigmoidal dose-response curves were seen at the clonal population level, but the activation thresholds for the individual T cells followed a Gaussian distribution. Moreover, the overall dose-response curve of the T cell clone revealed cyclic changes, becoming increasingly shifted toward lower Ag concentrations with the duration of time that elapsed since the last restimulation with Ag. Therefore, responsiveness to Ag (“functional avidity”) is not a constant parameter of a T cell clone but a function of the T cell’s history of last Ag encounter. The implications of such shifting activation thresholds are discussed for autoimmune disease.

High-resolution characterization of cytokine-producing alloreactivity in naive and allograft-primed mice.

BACKGROUND Whether alloreactive T cells in a naive host derive from naive or memory T cells remains unclear. It is also unclear whether graft rejection alters the phenotype of these T cells. Proliferation assays and cytokine enzyme-linked immunosorbent assays performed on culture supernatants do not differentiate primary T-cell alloreactivity from recall responses in allograft-primed mice, suggesting that these methods are inadequate measures of the alloreactive immune repertoire. METHODS To better characterize alloreactivity in naive and skin allograft-primed mice, we used a modified, high-resolution cytokine enzyme-linked immunosorbent spot assay capable of detecting cytokine production over short time periods. RESULTS Twenty-four-hour analysis of alloreactivity in mice that rejected fully MHC-disparate skin allografts revealed a high frequency of interferon (IFN)-gamma- and interleukin (IL)-4-producing, L-selectin-negative T cells, consistent with a memory phenotype. In contrast, 24-hr allostimulation of T cells from naive mice resulted in IL-2 production with minimal secretion of IFN-gamma or IL-4. The frequency of IL-2 producers was low and their phenotype was L-selectin positive, suggesting that they were naive and not memory T cells. When maintained in culture for 48 hr, however, the T cells from the primary mixed lymphocyte reaction began producing IFN-gamma, consistent with in vitro priming. CONCLUSIONS The primary alloresponse does not seem to involve clones that have been preprimed by environmental antigens, but instead behaves similarly to self-MHC-restricted immunity directed toward prototypic protein antigens: T cells with a naive phenotype are specifically induced to differentiate into high-frequency memory populations. These findings may have important implications for therapeutic induction of allograft tolerance.

T-cell epitope mapping using the ELISPOT approach

The ELISPOT assay is particularly well suited to measure both clonal size and effector function of low-frequency antigen-specific T-cell populations directly ex vivo. Typically, an ELISPOT assay is performed with a freshly obtained sample (or cryopreserved sample) using less than 24h of culture. Additionally, this assay allows for the simultaneous analysis of hundreds of variables in parallel from a single tissue specimen. Because of these capabilities, this assay has found widespread use in the context of direct ex vivo immune diagnostic monitoring in humans. Herein we describe the rationale, methodology, and useful hints for performing T-cell epitope mapping using ELISPOT analysis, and review typical results of such an analysis.