• CD4 T lymphocytes;
  • HIV;
  • flow cytometry;
  • history of medicine;
  • immunophenotyping gating strategies;
  • naive/memory T cells


  1. Top of page
  2. Abstract
  5. Acknowledgements

The story of T-lymphocyte subset immunophenotyping technology is reviewed on the occasion of the 20th anniversary of CD4 T-cell enumeration. Over time, immunophenotyping has evolved into precise, reliable, but complicated and expensive technology requiring fresh blood samples. The gating technologies that were universally adapted for clinical flow cytometry for the past decade relied on rapidly deteriorating morphological scatter characteristics of leukocytes. This special issue dedicated to CD4 T-cell enumeration features most of the available new options that will have a significant impact on how this technology will be implemented within the first decade of the 21st century. In a series of original publications, including the new NIH guideline for T-cell subset enumeration, contemporary gating protocols that use immunologically logical parameters are presented as part of the more reliable and affordable immunophenotyping alternative. Some of the improvements addressed here include the costs of the assays and the capacity to monitor interlaboratory and intralaboratory performances. It is clear that an effective attack on the human immunodeficiency virus (HIV) epidemic has to embrace resource-poor regions. Reducing the cost of the assay while improving reliability and durability is a move in the right direction. Cytometry (Clin. Cytometry) 50:39–45, 2002. © 2002 Wiley-Liss, Inc.

This volume of Clinical Cytometry is a commemorative issue to acknowledge and reflect on the 20-year battle with the human immunodeficiency virus (HIV) and to examine the distinct role of clinical cytometry in this struggle. This special issue is dedicated to three colleagues who lost their lives in the past year: Janis Giorgi, Mack Fulwyler, and Norman Jones. The launch of such a special issue can be compared with that of a launch of a sailboat for a voyage. Three elements are required: 1) a vision with energy to project the purpose and fascination for the voyage, 2) a compass to mark and stay the course, and 3) helmsmen to pilot the venture and to make sure it reaches the set goal (1). Such a scientific vision was inspired by Janis Giorgi, who instructed us how to fill the sails and begin the voyage (2). The compass, the trusted tool of old salts, was rendered by the great contributions of Mack Fulwyler and Norman Jones. Mack Fulwyler built the first sorting flow cytometer (3) and Norman Jones pioneered the way to visualize the binding of antibodies to cells (4,5). These have been pivotal contributions to navigate the uncharted waters of technology that took us from simple microscopy to tools of cytonomics. Finally, the contributors to this special issue are the helmspersons who keep their focus on the horizon, i.e., on our service commitments to provide effective patient monitoring and research in this protracted war against HIV.

This commemorative volume cannot be launched without a review of the past 20 years and without an assessment of our present activities. Future accomplishments in immunology will abound with countless new publications hopefully in the pages of this journal. Let this issue be the beginning of many successful copies that will lead to the eradication of this global plague.


  1. Top of page
  2. Abstract
  5. Acknowledgements

As if under divine inspiration, 20 years ago, the spirit of excitement and competitiveness among teams of cellular immunologists have “delivered the goods.” A diagnostic test was developed to enumerate CD4+ T lymphocytes using flow cytometry (6, 7). The very first clinical paper on “acquired (cellular) immunodeficiency” defined AIDS as a unique, newly emerging form of selective deficiency of “helper” CD4+ T lymphocytes (7). This was not just an amazing coincidence but also the result of a logical collaborative effort.

Test Systems

First, T-lymphocyte functional assays were needed in cultures of human blood in order to detect the potential and the heterogeneity of subpopulations. T-cell helper effects can be recognized by the induction of large-scale immunoglobulin secretion in activated B cells. A peculiar mitogen, pokeweed (PWM), known to stimulate both T and B cells in the mouse (8), was also found to activate human B cells. This stimulation in humans was T cell dependent, leading to a practical assay for analyzing helper T-cell function (9, 10) and complementing antigen stimulation tests and the mixed lymphocyte reaction (11).

Heterologous Antisera to T-Cell Subsets

It was Len Chess's insight that among heterologous antisera to human T lymphocytes, there was one, termed TH1, that reacted with some T cells (a subset, arbitrarily termed TH1+) that were functionally different from the rest of TH1- T lymphocytes (11). As some peripheral T-cell malignancies (T-chronic lymphocytic leukemia [T-CLL] and Sezary cells) showed uniform TH1+ cells, cells taken from such a malignancy were used to absorb antithymocyte sera in order to make a specific reagent to the complementary, so-called TH2+, T-cell subset (12). This procedure could be reproduced in different laboratories using rabbit as well as horse antithymocyte sera; 25%–35 % of blood T cells and >85 % of thymocytes were TH2+ (12, 13).

Monoclonal Antibodies to T-Cell Subsets

The first murine mAb to rat T cells, W3/25, was specific for the helper T-cell subset (14), proving the concept that mAbs can discriminate between lymphocyte subpopulations that show characteristic (in this case, helper) functions in vivo. The W3/25+ T-cell population showed helper cell activity for anti-hapten B cells and for graft-versus-host activity; W3/25- T cells mediated suppressive effects. Next, Stuart Schlossman's group collaborated with Patrick Kung and Gideon Goldstein at Ortho (Raritan, NJ; 15) to make a series of mAbs to human T cells, including OKT4 mAbs that reacted with 60%–70% of blood T cells and with >90% of thymocytes. The OKT4+ cells had helper function in the PWM assay and corresponded to the TH2- T cells. The OKT5 and OKT8 mAbs, on the other hand, reacted with TH2+ T cells that showed suppressor/cytotoxic features (16). Thus, a reagent set for the clinical analysis of immune disorders was assembled (7). At the same time, Bobby Evans with Becton-Dickinson (San Jose, CA) and soon Coulter (Miami, FL) also completed their mAb testing rounds, introducing Leu-3 and T4 as reacting with the OKT4+, TH2- T subset and Leu-2 and T8 as reacting with the TH2+, OKT8+, OKT4- T subset (6, 17). This “helper-suppressor/cytotoxic” dichotomy had a fundamental physiological significance. These subsets of T cells were clearly seeking different microenvironments in vivo (18). OKT4+(CD4+) T cells tended to seek the company of class II+ antigen-presenting dendritic cells and homed to germinal centers, whereas OKT8+(CD8+) T cells migrated to class I-rich sites such as the gut epithelium (18, 19). These subsets could be activated by different signals, associated with class II and class I molecules, respectively (17).

CD Numbers

In order to simplify the names of newly produced mAbs, leukocyte typing workshops (LTW) were organized (20). mAbs were grouped by the same CD number when reacted with the same CD (clusters of differentiation) antigens. At the first workshop in Paris in 1982, T4 and Leu-3a were included in the CD4 group and T8 and Leu-2a were placed in the CD8 group.

CD4 Is Part of the Receptor for HIV

Using this information, Klatzmann et al. (21) showed that each of the three CD4 mAbs tested blocked the infectivity of a lymphadenopathy virus (LAV). At the second workshop in Boston in 1984, Dalgleish et al. (22) used all 14 CD4 mAbs to show that each of these blocked the formation of multinucleated syncytia on mixing LAV or human T-cell lymphotropic virus-III (HTLV-III)– producing cells with CD4+ receptor-bearing cells. The remaining 140 mAbs enrolled at this workshop were ineffective. Thus, both studies concluded that the CD4 antigen was an essential and specific component of the receptor for the virus implicated in AIDS. The virus, referred to first as either LAV (23) or HTLV-III (24), was soon to be renamed HIV-1. Molecular analysis of the HIV envelope proteins and their interactions with the CD4 molecule followed (25). Next, the exact amino acid composition of the CD4 molecule, including the sites where the CD4 mAbs bind, was unraveled (26). These systems are among the best-studied molecular interactions in biology.

The True Significance: Saving Countless Lives

One wonders with awe what might have happened if the AIDS epidemic had appeared a mere 10 years earlier, in 1970, instead of later, around 1980, when both retrovirology (23, 24) and cellular immunology were already in full swing (6, 7). Even the most impartial observers of the current global AIDS scenario would feel a shiver down their spine while considering the increased severity of the worldwide HIV-related disaster that might have developed in the absence of these scientific discoveries. The HIV plague would have been further aggravated by prolonged confusion and wild speculation to cover up the ignorance about the cause of symptoms. An acknowledgment is in order to recognize CD4 immunologists for the breakthrough of the CD4 T-cell test. It was elegantly delivered at the time and at the place of need.


  1. Top of page
  2. Abstract
  5. Acknowledgements

By the mid 1980s, the unique trilogy fundamental to the successful execution of clinical flow cytometry had been implemented fully: 1) reliable, air-cooled argon ion lasers were mass produced, 2) modestly priced personal computers with astonishing capacities had become available, and 3) mAbs, produced in industrial quantities, were conjugated to a variety of fluorochromes (27). Currently, there are more than 20 methods requiring instruments with various levels of sophistication that can be implemented for CD4 T-cell enumeration (Table 1). Of these, T-cell subset enumeration by flow cytometry remains the undisputed choice for monitoring immune competence during antiretroviral therapy of HIV infection. The performance of laboratories that participate in HIV-related service is monitored regularly during quality assurance (QA) programs organized by the Centers for Disease Control (CDC), the National Institutes of Health (NIH), College of American Pathologists (CAP), Quality Assessment and Standardization for Immunological Measures Relevant to HIV/AIDS (QASI), and the United Kingdom National External Quality Assessment Schemes for Immune Monitoring (UK NEQAS), and frequently also by the commercial flow cytometry companies.

Table 1. Methodologies Compatible with CD4 T-Cell Enumeration
Operating principleDescription of instrumentDescription of processingSystem model nameManufacturer
  • a

    Has not been marketed as a CD4 T-cell counting system.

Cytometry-based methods
 FlowBenchtopVolumetricCytoronAbsoluteOrtho Diagnostics
 FlowBenchtopFlow rateCaliburBecton Dickinson
 FlowBenchtopFlow rateFACScanBecton Dickinson
 FlowBenchtopFlow rateEpics XLBeckman Coulter
 FlowBenchtopFlow rateProfile IIBeckman Coulter
 FlowCompactUniversalLuminex 100aLuminex
 FlowCompactDedicatedFACSCountBecton Dickinson
 FlowHematology analyzerUniversalImmuno VCSBeckman Coulter
 FlowHematology analyzerUniversalCellDyn 4000Abott
 StaticScanningImmunomagneticCell TracksImmucore
 StaticCapillarySpatialImagn2000Beckton Dickinson
Noncytometry-based methods
 Cell membraneSolid phaseMagnetic beadsCapcelliaBioRad
 Cell membraneMicroscopyMagnetic beadsDynabeadsDynal
 Cell membraneSolid phaseMagnetic beadsZymmune
 Cell membraneSolid phaseMagnetic beadsMACSMilyenyi
 Cell membraneMicroscopyRosetting beadsCytospheresBeckman Coulter
 Soluble proteinSolid phaseDirectTRAxInnogenetics
 Soluble proteinSolid phaseIndirectImmunoDiagnosticsImmuno Diagnostics

Most reviews (28) and several papers in this issue (29–31), as well as the experience with various QA programs, indicate that the CD4 T-cell results can be obtained with high precision provided that the tests are performed on fresh blood preparations. There are, however, two significant problems in the field of immunophenotyping that are pertinent for further considerations. First, in the past, the technology had little flexibility or “reserve” power for analyzing samples that had been handled suboptimally. Conventional leukocyte subset delineation, based on morphological scatter characteristics, shifted with the increasing time of storage of the specimen. Therefore, reliable lymphocyte identification with high purity and recovery was possible only from freshly prepared blood. The long standing challenge—how to obtain both high purity and recovery from blood that is more than 24 hours old—is addressed in this issue (29, 32). Second, current protocols appeared to be complicated renditions of protocols derived from experience associated with research investigations. They provided results, in addition to CD4 T cells, that were frequently not requested nor required by clinicians. Both problems are derived from the blind adherence to the “established” complicated methods. Gating exclusively on intrinsic attributes of cells prevented the widescale implementation of more robust and cost-effective flow cytometric options. Until now, none of the optional methods were able to supersede the entrenched technology reigning since the early 1980s (29). The new data presented in this issue show that the monopoly of the traditional immunophenotyping approach is fading in a convincing fashion.

Historically, modern immunophenotyping technology goes back to the initial mass-produced instruments of the late 1970s. They were able to detect with a single laser two morphological or intrinsic cell attributes as initial parameters. At that time, fluorescence detection was limited to a single color. The morphology-based parameters were orthogonal and forward light scattering with one other parameter available on some instruments. It was cell volume based on electrical impedance. The cell volume feature was short lived and by the mid 1980s had disappeared from clinical instruments. The initial insistence by cell biologists on morphological parameters was justified, as fluorescent detection at that time was providing limited information for three reasons. First, there was a limited knowledge about leukocyte surface differentiation biology; most receptors were yet to be discovered. Second, it was laborious to produce lineage and subset-specific heteroantisera by extensive absorption protocols. Third, there were serious limitations regarding fluorescent dyes that were compatible with the excitation wavelength of the argon ion laser. Consequently, designing engineers placed considerable emphasis on the image pattern recognition skills of pathologists. Image pattern recognition was attractive. Indeed, technologists in these laboratories were able to recognize cell clusters based on bivariant dot plots derived from composite morphological features. This marketing strategy, well received by experts trained in aspects of morphological analysis, was apparently successful. In hospital laboratories, flow cytometers gradually replaced the epifluorescent microscopes. There was a sevenfold increase in flow cytometer numbers globally from the mid 1980s to the mid 1990s (33). This homogeneous gating approach, based on the intrinsic morphological scatter features of the cells, persisted for 20 years as the primary, first-to-be-used, gating strategy.

It is remarkable how well such a troubled, morphologically based, and vulnerable protocol was maintained, considering that from the mid 1970s the concept of triggering on fluorescence was well known. The acronym, fluorescent activated cell sorting (FACS), precisely describes that the cells are investigated primarily for their immunofluorescence features. Yet, this practical and immunologically logical concept for immunophenotyping analysis was essentially ignored for two decades. Tolstoy in his great novel, Anna Karenina, stated: “Happy families are all alike; and every unhappy family is unhappy in its own unique way.”1 The implication is that challenges to historical well-established precedents are not to be taken seriously. The issues revolve around two simple practical considerations. First, a wide range of mAbs with exquisitely precise specificity and conjugated to a variety of fluorochromes are available. Second, these markers of cellular functions are preserved better on cells, for longer periods, than the morphological scatter features of these populations. It was not until the mid 1990s that immunologists began to take advantage of lineage-specific markers as powerful primary gating tools. The utility of the CD3 and CD45 mAbs was published in 1992 and 1996, respectively (34, 35). However, there was little impact from these findings. Then, the ImmunoCount protocol on the Ortho CytoronAbsolute defined lymphocytes, primarily the sum of CD3+ T, CD19+ B, and CD16+ natural killer (NK) cells (36), and the primary gating of CD34+ precursor cells was also explored.

The conclusion is, therefore, that in modern practical protocols of flow cytometry, it is advantageous to abandon the first use of homogeneous morphological discrimination using the display of forward scatter (FSC) versus orthogonal side scatter (SSC) on the x and y-axes. Instead, it is more practical to adopt the heterogeneous “morphospectral” strategy for primary gating where (at least) one parameter is based on the reactivity of a fluorochrome-conjugated antibody (e.g., CD45-fluorescein isothiocyanate [FITC] or CD3-phycoerythrin [PE]-Cy5) used together with the SSC (29, 30, 34, 35). Similarly, a heterogeneous display of CD4-FITC and SSC on the y and x-axes represents a direct CD4 gating strategy (37). Importantly, the momentum for a change in immunophenotyping is now rapidly growing. This change is being facilitated by the arrival of the powerful generic antiretroviral drugs to the resource-poor areas of the world where they are needed the most. To date, cost-effective implementation of T-cell subset monitoring technology has eluded these regions. As a worldwide effort, simpler, better, and eventually cheaper tests for T-cell subsets are being developed (32, 37–41). From the evidence presented in this issue, it appears that with single-platform absolute count technologies that incorporate CD45 gating, an optimal package has arrived (29, 37, 40–42). O'Gorman and Nicholson (38) discussed the advantages of single- platform technology. Both the new protocol and its utility are covered in this issue (32, 38). Another alternative method, a CD45 gating protocol with dual platform absolute count technology referred to as “panleukogating,” is also presented in this issue (30). There is also a volumetric absolute count solution. It avoids the complications and the expense of the fluoromicrosphere-based absolute counting and can be married with the use of CD45 and SSC in combination with a single function-specific marker, CD4 (39). Both the CD45 and CD4 reagents are available as generic mAbs (41) to resource-poor countries, presenting these countries with affordabable, simple, and robust technology, along with the concepts of the heterogeneous morphospectral gating combination as described above.

Finally, it is interesting to note that the combinations of intrinsic and extrinsic cell attributes, the salient features of optimal primary gating, were first conceptualized by Shapiro (43) in flow cytometry. Nevertheless, the general application of this concept of intrinsic versus extrinsic attributes of cells and microbes goes back to Paul Ehrlich who, in 1882, worked in Robert Koch's laboratory in Berlin. Koch had just identified the tuberculosis bacilli, and Ehrlich developed the technique for staining it. Ehrlich's motto was “Corpora non agunt nisi fixata” or “Bodies do not act unless fixed” (44). Translated to the jargon of flow cytometry, Ehrlich's motto means “cells do not reveal their lineage affiliation and functional roles in immunity unless labeled by mAbs directed to the relevant functional differentiation antigens.” Ehrlich had all the intuition and energy to probe human tissues with dyes, heat, and chemical means to extract extrinsic cellular information. He probably would have enjoyed to see that his sketches of lymphocytes carrying a multiplicity of hypothetical cell surface receptors are still popular among medical students 100 years after having been drawn (45) and would have been impressed that we can visualize these receptors by flow cytometry. Of course, Paul Ehrlich, the old visionary, did not get everything totally right. Therefore, there is ample room left to study cell-to-cell interactions in the 21st century.

Monitoring Immune Status

In this review, most of the focus has been on the general surrogate marker for monitoring the immune system of HIV patients. Although the simultaneous three and four-color capacity of contemporary flow cytometers may not be utilized fully for basic T-cell subset enumeration, they are required for more sophisticated monitoring of immune status. As more effective antiretroviral combinations are discovered so are new immunophenotyping markers. They provide more direct information about the immune status of individuals living with HIV and how they respond to treatment. In 1995, a new era began when new antiretroviral drug regimens combined the inhibitors of two HIV enzymes, reverse transcriptase (RTI) and protease (PI). These regimens induced major and stable reduction in viral load accompanied by sustained CD4 cell increases (46–49) that had never been reported with monotherapy or bitherapy with RTI. These changes immediately raised the question of immune restoration and its mechanisms. A very early increase in CD4 T-cell counts was characterized by strong slopes of peripheral blood CD4 T cells of 1–5 CD4 cell/mm3 per day during the first 2 months of treatment concurrently with a rapid and major reduction in virus load of 1 or 2 logs of magnitude.

The kinetics of CD4 cell expansion is usually reduced after the second or third month of treatment (50–53). Simple flow cytometry analyses helped to demonstrate the mechanisms of CD4 T-cell reconstitution. In the first study of its kind, the rapid early wave of CD4 T cells was composed mostly of memory CD4 T cells bearing the CD45RO isoform. Regeneration of naive T cells played a major role in the second phase of the CD4 T-cell subset reconstitution. Indeed, naive CD4 T cells significantly increased both in proportion and absolute number after the third month of treatment in patients treated in the late stages of CD4 T-cell depletion. These first findings were confirmed by several subsequent studies (51, 52, 54). However, when treatment is introduced at earlier stages of the disease, or even at primary infection (55), the slopes of naive CD4 T-cell recovery are indistinguishable from the memory CD4 T cells. A rapid and early increase of naive CD4 T cells indicates that the naive T cells can be submitted as memory T cells, to some sequestration/desequestration phenomenon. After the first 3–6 months of treatment, the slopes of naive cell recovery appear to be parallel, independent of the disease stage, suggesting a very conserved mode of replenishment of the CD4 T-cell compartment. In the long-term, naive T-cell reconstitution is the main component of the total CD4 T-cell reconstitution.

This increase in naive T cells was the major surprise. When one considers the dogma of a thymic involution in adults, derived from observations of late naive T-cell regeneration in adults after chemotherapy for hematological malignancies (61, 62), such an increase in naive T-cells was a major surprise. These flow cytometry-based observations immediately raised a controversy about the origin of these naive CD4 T cells. Indeed, some memory T cells can revert their CD45 isoform from RO to RA (58), although such a phenomenon occurs mostly in the CD8 T-cell compartment. The naive T-cell status was assessed further by demonstrating the coexpression of CD45RA and CD62L-selectin (50). The latter molecule allows naive T-cell penetration in lymph nodes (59). Also, it was established that CD45RA+62L+CD4+ cells had the functional characteristics of naive T cells that had not encountered antigens and were unable to either proliferate in response to recall antigens or produce effector cytokines. These results were confirmed by Pakker et al. (51) and Lederman et al. (52), further strengthening the hypothesis of a preserved capacity to regenerate naive T cells during HIV infection in adults. Direct evidence of thymic participation and T-cell regeneration during antiretroviral therapy came from the molecular detection of markers of thymic origin in the same subset of naive T cells (60–62). In the subset of immunophenotypically defined CD45RA+CD62L+CD4 T cells, it is possible to find naive CD4 T cells that have not undergone multiple cycles of proliferation after leaving the thymus. A proportion of the T-cell receptor (TCR) rearrangement excision circles (TRECs) are one tenth of the immunophenotypically defined naive CD4 T cells that harbor TRECs (Douek). Thus, the relative proportions of TRECs within the CD4 T-cell subsets fit with the phenotypic definition of naive and memory T cells.

With this brief journey into the state of our understanding of the subdivision among T-cell subsets, it is clear that multiparameter and multicolor flow cytometry will help to navigate the challenging voyages into cytonomics.


  1. Top of page
  2. Abstract
  5. Acknowledgements

This work was supported in part by grant QLRI-2000-00436“Quality of Life and Management of Living Resources” from the European Commission, Brussels, Belgium.

  • 1

    The quote is from a French translation of L. Tolstoy by Henri Mongault from “Anna Karénine,” Le Livre de Poche, Paris, 1960. p. 1. Translated from French to English by F. Mandy. The original sentence in French is: “Les families heureuses se ressemblent toutes; les familles malheureuses sont malheureuses chacune à leur facon.”


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