Louis J. Picker

Lymphocyte homing and homeostasis

The integration and control of systemic immune responses depends on the regulated trafficking of lymphocytes. This lymphocyte “homing” process disperses the immunologic repertoire, directs lymphocyte subsets to the specialized microenvironments that control their differentiation and regulate their survival, and targets immune effector cells to sites of antigenic or microbial invasion. Recent advances reveal that the exquisite specificity of lymphocyte homing is determined by combinatorial “decision processes” involving multistep sequential engagement of adhesion and signaling receptors. These homing-related interactions are seamlessly integrated into the overall interaction of the lymphocyte with its environment and participate directly in the control of lymphocyte function, life-span, and population dynamics. In this article a review of the molecular basis of lymphocyte homing is presented, and mechanisms by which homing physiology regulates the homeostasis of immunologic resources are proposed.

Changes in thymic function with age and during the treatment of HIV infection

The thymus represents the major site of the production and generation of T cells expressing αβ-type T-cell antigen receptors. Age-related involution may affect the ability of the thymus to reconstitute T cells expressing CD4 cell-surface antigens that are lost during HIV infection; this effect has been seen after chemotherapy and bone-marrow transplantation,. Adult HIV-infected patients treated with highly active antiretroviral therapy (HAART) show a progressive increase in their number of naive CD4-positive T cells,. These cells could arise through expansion of existing naive T cells in the periphery or through thymic production of new naive T cells,. Here we quantify thymic output by measuring the excisional DNA products of TCR-gene rearrangement. We find that, although thymic function declines with age, substantial output is maintained into late adulthood. HIV infection leads to a decrease in thymic function that can be measured in the peripheral blood and lymphoid tissues. In adults treated with HAART, there is a rapid and sustained increase in thymic output in most subjects. These results indicate that the adult thymus can contribute to immune reconstitution following HAART.

HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression.

The role of HIV-1-specific CD4+ T-cell responses in controlling HIV-1 infection remains unclear. Previous work has suggested that such cells are eliminated in the early stages of infection in most subjects, and thus cannot substantially contribute to host defense against HIV-1. Here, using flow cytometric detection of antigen-induced intracellular cytokines, we show that significant frequencies of gag specific, T-helper-1 CD4+ memory T cells are detectable in most subjects with active/progressive HIV-1 infection (median frequency, 0.12% of memory subset; range, 0–0.66%). Median frequencies of these cells were considerably higher in nonprogressive HIV-1 disease (0.40%), but there was substantial overlap between the two groups (range of nonprogressors, 0.10–1.7%). Continuous HIV-1 suppression with anti-retroviral therapy was associated with a time-dependent reduction in median frequencies of gag-specific CD4+ memory T cells: 0.08% in subjects treated for 4–24 weeks, and 0.03% in subjects treated for 47–112 weeks. Thus, functional HIV-1-specific CD4+ T cells are commonly available for support of anti-HIV-1 effector responses in active disease, but their decline with anti-retroviral therapy indicates that immunologic participation in long-term HIV-1 control will probably require effective vaccination strategies.

Determination of antigen-specific memory/effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency.

The highly regulated secretion of effector cytokines by CD4+ T cells plays a critical role in immune protection against pathogens such as cytomegalovirus. Here, we directly compare the frequency and functional characteristics of cytomegalovirus-specific CD4+ memory/effector T cells in normal and HIV+ subjects using a novel, highly efficient multiparameter flow cytometric assay that detects the rapid intracellular accumulation of cytokine(s) after short-term (6 h) in vitro antigen stimulation. Responses in this assay correlate precisely with independent measures of sensitization history (e.g., seroreactivity), and allow the simultaneous assessment of multiple cytokines in single effector T cells. Healthy HIV- individuals manifested an average of 0.71, 0.72, 0.38, and 0.06% CD4+ T cells responding to cytomegalovirus with gamma-IFN, TNF-alpha, IL-2, and IL-4 production, respectively, with the simultaneous production of gamma-IFN, TNF-alpha, and IL-2 being the most common effector phenotype. Significantly, overall cytomegalovirus-specific CD4+ effector frequencies were markedly higher among 40% of HIV+ subjects (2.7-8.0%), and demonstrated a predominately polarized gamma-IFN+/TNF-alpha+/IL-2-/IL-4- phenotype. In contrast, CD4+ effector frequencies for heterologous, nonubiquitous viruses such as the mumps virus were low or absent in the HIV+ group. These data suggest the existence of homeostatic mechanisms in HIV disease that selectively preserve memory T cell populations reactive with ubiquitous pathogens such as cytomegalovirus-likely at the expense of T cell memory to more sporadically encountered infectious agents.

Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry

We have recently described a highly sensitive flow cytometric technique, based on the ability to detect single cell expression of cytokines, to simultaneously quantitate and phenotypically characterize antigen-specific memory/effector T cells in PBMC cultures. In this report, we describe a simplified procedural modification which enables the rapid detection of low frequency memory CD4+ and CD8+ T cells expressing cytokines in response to soluble antigen in whole blood. When compared with T cell responses in PBMC cultures, whole blood cultures demonstrated similar but slightly higher percentages of T cells responsive to specific antigen. In addition, T cell responses to cytomegalovirus in whole blood were observed only in sensitized (seropositive) individuals, and CD4+ T cell responses could be blocked by anti-class II MHC antibodies. This procedure may provide a means to examine direct effects of pharmacological drug concentrations on T cell immunity in clinical samples.

Homing Receptors and Vascular Addressins: Cell Adhesion Molecules that Direct Lymphocyte Traffic

The trafficking of lymphocytes and other leukocytes plays a major role in the physiology of the itnmune system and the implementation of immune mechanisms. The recirculation of lymphocytes allows the full repertoire of antigenic specificities to be continuously represented throughout the body, and may also facilitate interactions between lymphocyte subsets and accessory cells required for antigen-specific effector functions. In addition, lymphocyte trafficking plays a role in segregating particular effector and memory subpopulations permitting the development of unique responses in, for example, mucosal versus non-mucosal sites. The selective extravasation of other classes of leukocytes, such as monocytes and neutrophils, contributes to inflammatory reactions resulting in the destruction of foreign microorganisms, clearance of tumors and the expulsion of parasites. The first step reqtiired for lymphocyte entry into peripheral lymph nodes and mucosal lymphoid organs (Peyers patches and appendix) from the blood is the recognition and adhesion to postcapillary venules lined by high endothelium (HEV). Lymphocytes as well as HEV employ specific cell surface molecules for this function and these or related molecules are also important in directing the extravasation of other leukocytes. In this manuscript we will briefly outline our view of the role of lymphocyte-HEV interactions in lymphocyte traffic, and will summarize our studies of some of the molecules involved. We will discuss the

Identification of Functional Subsets by Flow Cytometry: Intracellular Detection of Cytokine Expression

Methods for analysis of T cell function have traditionally relied upon measurements of proliferation or cytokine expression in bulk cultures of PBMC in long term incubations with polyclonal mitogens or putative antigen. These techniques suffer from the drawback that they do not enable analysis of single cell responses in the context of unselected cellular backgrounds. In addition these methods are not sensitive enough to rapidly assess rare event responses characteristic of cognate memory T cell responses. This review discusses recently developed flow cytometric methods designed to rapidly assess leukocyte subset cytokine responses to polyclonal activators and specific antigen in PBMC and whole blood samples. These procedures determine the percentages of activated cells and the identification of leucocyte subsets capable of expressing various cytokines and cell surface antigens. The ability to assess key intracellular functional markers by multiparameter flow cytometry offers some unique advantages in a number of clinical applications. The technical simplicity and rapidity of the flow cytometric intracellular cytokine detection techniques described in this report, as well as the widespread availability of appropriate flow cytometers and cell surface directed antibodies in clinical laboratories, suggests the possibility that this technique could be broadly applicable to the clinical evaluation of immune status. Since any cell type can be identified with this approach, responses to a variety of clinically relevant stimuli in virtually any leukocyte subset can be evaluated including monocyte responses to LPS, and T cell responses to mitogens and a variety of bacterial and viral antigens. The significance of measuring low frequency antigen-specific responses with respect to clinical significance in assessing immune status in a variety of clinical conditions and determining efficacy or immunotoxicity of drugs and vaccine antigens is discussed.

Putative Immunodominant Human Immunodeficiency Virus-Specific CD8+ T-Cell Responses Cannot Be Predicted by Major Histocompatibility Complex Class I Haplotype

ABSTRACT Recent studies of human immunodeficiency virus (HIV)-specific CD8+ T cells have focused on responses to single, usually HLA-A2-restricted epitopes as surrogate measures of the overall response to HIV. However, the assumption that a response to one epitope is representative of the total response is unconfirmed. Here we assess epitope immunodominance and HIV-specific CD8+ T-cell response complexity using cytokine flow cytometry to examine CD8+ T-cell responses in 11 HLA-A2+HIV+ individuals. Initial studies demonstrated that only 4 of 11 patients recognized the putative immunodominant HLA-A2-restricted p17 epitope SLYNTVATL, suggesting that the remaining subjects might lack significant HIV-specific CD8+ T-cell responses. However, five of six SLYNTVATL nonresponders recognized other HIV epitopes, and two of four SLYNTVATL responders had greater responses to HIV peptides restricted by other class I alleles. In several individuals, no HLA-A2-restricted epitopes were recognized, but CD8+ T-cell responses were detected to epitopes restricted by other HLA class I alleles. These data indicate that an individuals overall CD8+ T-cell response to HIV is not adequately represented by the response to a single epitope and that individual major histocompatibility complex class I alleles do not predict an immunodominant response restricted by that allele. Accurate quantification of total HIV-specific CD8+ T-cell responses will require assessment of the response to all possible epitopes.

A two-step adhesion cascade for T cell/endothelial cell interactions under flow conditions.

Neutrophil adherence to endothelial cells (ECs) under conditions of flow occurs in successive steps, including selectin-dependent primary adhesion and CD18-dependent secondary adhesion. We used a parallel-plate flow chamber to assess the steps in T cell adherence in vitro. On monolayers of L cells transfected with the EC adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1), or intercellular adhesion molecule-1 (ICAM-1), E-selectin was capable of mediating only primary adhesion, ICAM-1 was capable of mediating only secondary adhesion, and VCAM-1 was capable of mediating both primary and secondary adhesion. Studies using human umbilical vein EC monolayers stimulated for 24 h with IL-1 also revealed distinct primary and secondary steps in T cell adhesion under flow, and the secondary adhesion was inhibited > 90% by blocking both VCAM-1/alpha 4 beta 1 integrin and ICAM-1/CD18 integrin pathways. However, the primary adhesion under conditions of flow could not be attributed to any of the mechanisms known to support adhesion of leukocytes to ECs. Alone, this pathway was shown to mediate T cell rolling and was a necessary prerequisite for engagement of the two integrin pathways in this system. Thus, T cell adherence to 24-h IL-1-stimulated human umbilical vein ECs at venular wall shear stresses involves at least two successive steps, with clear molecular distinctions from the mechanisms accounting for neutrophil/EC adhesion.

Distribution of human CMV‐specific memory T cells among the CD8pos. subsets defined by CD57, CD27, and CD45 isoforms

Chronic antigenic stimulation has been associated with peripheral blood expansions of CD8pos. T cells characterized by CD57 expression, loss of CD27 expression, and reversal of the CD45RObright /RAdim phenotype usually associated with immunological memory towards a CD45ROdim /RAbright phenotype. However, the relationship and functional significance of these subset(s) has remained controversial. Here, this issue was addressed using a novel flow cytometric technique that allows simultaneous detection of human cytomegalovirus (HCMV)‐specific CD8pos. memory T cells by rapid (< 6 h) HCMV peptide‐specific induction of cytokine synthesis, and their phenotypic characterization, including CD57, CD27 and CD45RA/RO. The vast majority of resting CD8pos. T cells capable of rapid induction of IFN‐γ and TNF‐α synthesis in response to HCMV peptides were found in a subset characterized by intermediate to high expression of CD57, down‐regulation/loss of CD27, and varying degrees of reversal of the classical “memory” CD45RObright /RAdim phenotype. This subpopulation likely includes the fully differentiated memory cells responsible for the long‐term immune defense against HCMV reactivation.