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Keywords:

  • HIV;
  • AIDS;
  • cytometry;
  • immune system

Abstract

  1. Top of page
  2. Abstract
  3. The Epidemics
  4. Changes in CD4+ T Cells, the Main Target of HIV, and in Their Subtypes Have Been Identified by Using FCM
  5. The Importance of T-Cell Activation and CD4+ T-Cell Subtypes
  6. CD8 T Cells and HIV Infection
  7. Natural Killer Cells and HIV Infection
  8. Alterations in the B-Cell Compartment
  9. The Mononuclear Phagocytic System
  10. A Space for FCM Analysis of Soluble Molecules
  11. Conclusions
  12. Literature Cited

Flow cytometry (FCM) has been extensively used to investigate immunological changes that occur from infection with the human immunodeficiency virus (HIV). This review describes some of the most relevant cellular and molecular changes in the immune system that can be detected by FCM during HIV infection. Finally, it will be discussed how this technology has facilitated the understanding not only of the biology of the virus but also of the mechanisms that the immune system activates to fight HIV and is allowing to monitor the efficacy of antiretroviral therapy. © 2013 International Society for Advancement of Cytometry


The Epidemics

  1. Top of page
  2. Abstract
  3. The Epidemics
  4. Changes in CD4+ T Cells, the Main Target of HIV, and in Their Subtypes Have Been Identified by Using FCM
  5. The Importance of T-Cell Activation and CD4+ T-Cell Subtypes
  6. CD8 T Cells and HIV Infection
  7. Natural Killer Cells and HIV Infection
  8. Alterations in the B-Cell Compartment
  9. The Mononuclear Phagocytic System
  10. A Space for FCM Analysis of Soluble Molecules
  11. Conclusions
  12. Literature Cited

With no presumption of being exhaustive, this review briefly describes how flow cytometry (FCM) has been used in the last 30 years to investigate the effects of human immunodeficiency virus type-1 (HIV-1) infection on the immune system. Main observations and key points will be presented and discussed, underlying the importance of cytometric approaches. We apologize to all those scientists whose work and discoveries cannot be quoted here for lack of space.

The HIV/AIDS epidemics has an official birth date, which is June 1981, when the Centers for Disease Control's Morbidity and Mortality Weekly Report evidenced a cluster of five cases of lethal pneumonia due to Pneumocystis jirovecii in the area of Los Angeles, further described in a pivotal paper that was published in December 1981 ([1]). After more than 30 years, the HIV is still causing highly dramatic epidemics, whose dimension, in terms of infected patients and victims (http://www.unaids.org), is not decreasing as many scientists were expecting. HIV infection has indeed spread worldwide, leaving no region of the world unaffected, and still represents a major challenge for physicians and scientists, as well as economists and politicians.

If untreated, the infection provokes an inexorable and catastrophic decline in the quality of immune responses, which is based on the destruction of CD4+ T lymphocytes, and then involves almost all of the cells and mechanisms responsible for the immune protection of the body. Effective drugs exist, collectively defined as “highly active antiretroviral therapy” (HAART) that, when adequately used and with precise strategies, in most cases block HIV production and activities at various steps, from cell entry to the final assembly of the virus, therefore keeping the infection dormant even for extremely long periods ([2]). It is to note, however, that at present and with the current weapons, the virus cannot be eradicated from patients, and even in those who seem to control the infection signs of its presence, well evidenced by the analysis of immune activation markers, can be found ([3]).

The HIV scenario, at present, remains frankly tragic: most infected persons worldwide are not treated, or receive an incomplete treatment, even if drugs can be produced at a relatively low cost and could be easily shipped almost everywhere. Several public health programs and many large therapy projects that had started years ago have then failed, and a vaccine is still very far away. As a consequence, millions of HIV+ persons suffer from dramatic immune alterations, and thousands of them perish without any medical help, without knowing which infection is affecting them and what could have been done to prevent their disease.

On the other hand, patients who have the possibility to assume HAART have to be treated for the rest of their life and can develop drug-related side effects. This can lead to an altered assumption of antiretroviral therapies, further favoring the epidemics. In some patients, viral strains that are resistant to one or more drugs or even to one or more drug categories can emerge. Moreover, the fact that effective therapies have decreased morbidity and mortality has paradoxically caused a relaxation of adequate public and private health measures that threaten a recrudescence of the epidemics.

In such a scenario, where economic and political interests also play a role, scientists have always been working with unprecedented efforts and devotion to the cause and have developed a number of tools, technologies, and strategies to understand the biology of HIV infection and its effects on the immune system. Among these, FCM has played from the beginning a crucial, if not unique, role. Indeed, the necessity to identify and quantify CD4+ T cells has pushed the development of new and more friendly instruments. The first flow cytometers used for this purpose were extremely complex and quite difficult to use, were equipped with potent lasers that required a high amount of cold water for cooling, and typically had the possibility to detect two physical parameters along with two fluorescences. Then, after 1985, a period that could be defined “CD4cene” (the cytometric equivalent of Holocene, the geological epoch in which we are living) started: to cover the need of performing a high number of CD4+ T cell counts because of the expansion of HIV epidemics, the market saw the arrival of the first nonsorting benchtop flow cytometers that were equipped with air-cooled lasers and whose cost was much more affordable than that of previous instruments. They had a great economic success, being capable to join clinical and research requirements, and, after 3–4 years, also gave the possibility to analyze a third fluorescence, followed by the fourth when a second laser could be added.

Changes in CD4+ T Cells, the Main Target of HIV, and in Their Subtypes Have Been Identified by Using FCM

  1. Top of page
  2. Abstract
  3. The Epidemics
  4. Changes in CD4+ T Cells, the Main Target of HIV, and in Their Subtypes Have Been Identified by Using FCM
  5. The Importance of T-Cell Activation and CD4+ T-Cell Subtypes
  6. CD8 T Cells and HIV Infection
  7. Natural Killer Cells and HIV Infection
  8. Alterations in the B-Cell Compartment
  9. The Mononuclear Phagocytic System
  10. A Space for FCM Analysis of Soluble Molecules
  11. Conclusions
  12. Literature Cited

The first paper reporting changes in the absolute number of peripheral blood CD3+ (at that time, named T3+ lymphocytes), CD4+, and CD8+ T cells was published in 1983 and regarded a study on 15 patients with a unknown, new form of immune deficiency ([4]). FCM was used to demonstrate that, in comparison with healthy controls, they had a lower number of CD4+ cells and a lower CD4/CD8 ratio. At present, the number of scientific papers that have somehow analyzed CD4+ cells (including monocytes that are CD4low) during HIV infection is impressive. According to PubMed, in 1984 (when CD4 antigen was named T4), there were less than 10 publications describing the loss of such cells during a new form of immunodeficiency. In 2012, papers on this topic were more than 2,500 and, as of May 2013, their total number is much higher than 40,000.

As far as the immunopathogenesis of the infection is concerned, the pivotal observation that a subset of T lymphocytes positive to a surface glycoprotein with a molecular mass of 62 kDa termed “T4” (i.e., CD4+ T lymphocytes) was depleted in patients with persistent generalized lymphadenopathy arrived 30 years ago ([4]). From that moment, and until the arrival of the quantification of plasma viral load (the first test was developed by Roche, the Amplicor HIV-1 Monitor Test, FDA approved in February 1999), the count of CD4+ T cells by FCM was the only marker of the severity of the infection and of the efficacy of the treatments. It is to note that, nowadays, almost all clinical papers do not report how the number of CD4+ T cells is calculated, taking for granted that FCM is used for this purpose.

The count of CD4+ T cells, and eventually the analysis of lymphocyte phenotype, is still crucial for monitoring the infection and its treatment. Several panels based on different mAbs (up to six, for routine analysis) and numerous FCM approaches are now available that make use of a number of markers for the precise identification of such cells, typically performed excluding cells with different surface molecules. It is logical that, in principle, the perfect identification of a given population, positive to one or two markers and negative to many others, is of interest. However, it has to be noted that sometimes CD4+ T cell count is performed with an excessive use of reagents and on highly expensive instruments, with a cost that is becoming unaffordable even in western countries, where resources for medical care are constantly decreasing. CD4 molecule is well expressed on T-helper lymphocytes, and it has been shown by several groups, including ours, that even the use of one single anti-CD4 antibody, conjugated with a brilliant fluorochrome, is sufficient for measuring this parameter and to give a correct count that can be used in the clinical practice (manuscript submitted). Thus, a Ferrari can be used to deliver the mail; however, there are more economical cars that can perform the same function.

Apart from the problem related to their count, CD4+ T cells have been largely studied to identify, among them, those types and functions that are mostly affected by the infection. Several pivotal observations were thus made on the importance of T-cell subsets, and it was found that three main phases exist in the reconstitution of the CD4+ T-cell pool after giving patients HAART ([5]). In the first phase, an early rise of memory CD4+ T lymphocytes takes place, followed by a reduction in T-cell activation and an improved response to recall antigens, and finally, a late increase of virgin, newly formed naïve CD4+ T cells.

The use of markers related to virgin or memory cells also allowed to identify a crucial mechanism explaining the loss of virus-specific CD4+ T lymphocytes ([6]). Indeed, it was found that in all stages of the infection, memory CD4+ T cells that were specific for the virus contained more HIV DNA than other memory cells and that treatment interruptions caused an increase in HIV DNA that was much greater in HIV-specific cells. Thus, use of markers allowed to show that HIV-specific CD4+ T cells are preferentially infected by HIV.

Studies on of the Vβ-TCR repertoire, detecting the clonality of different Vβ families by FCM and spectratyping, revealed that before the beginning of the therapy, the repertoire of patients with acute or chronic infection was significantly different from that of healthy controls ([7]). Patients with acute HIV infection showed an improvement of the repertoire among either CD4+ or CD8+ T lymphocytes after successful HAART. Those with chronic infection were capable of changing their repertoire among CD8+ but not CD4+ T cells, suggesting that therapy cannot restore the T-cell repertoire in individuals whose immune system is already severely compromised by the infection.

The Importance of T-Cell Activation and CD4+ T-Cell Subtypes

  1. Top of page
  2. Abstract
  3. The Epidemics
  4. Changes in CD4+ T Cells, the Main Target of HIV, and in Their Subtypes Have Been Identified by Using FCM
  5. The Importance of T-Cell Activation and CD4+ T-Cell Subtypes
  6. CD8 T Cells and HIV Infection
  7. Natural Killer Cells and HIV Infection
  8. Alterations in the B-Cell Compartment
  9. The Mononuclear Phagocytic System
  10. A Space for FCM Analysis of Soluble Molecules
  11. Conclusions
  12. Literature Cited

It is well known that a persistent immune activation is a hallmark of HIV infection, can predict disease progression independently of viral load, and is typically present during acute, primary infection when most activated cells show a high tendency to undergo apoptosis ([8]). The importance of this phenomenon has been recently remarked by the observation that the level of activated cells can predict the capacity to control viral infection and thus the length of the therapy-free period, even from the first phases of the infection, as revealed by studies during primary, acute infection ([9, 10]). Several markers have been used to identify activated T cells, including CD38, CD95, and HLA-DR, possibly combined ([9, 11]), along with the quantification of the expression of several cell adhesion molecules, among which are integrins.

Other types of CD4+ T cells are under investigation, including CD4+ natural regulatory cells (Tregs), which suppress antigen-specific T-cell responses and reduce the hyperactivation of the immune system. Their role and influence during HIV infection remains unclear. Two main reasons could explain this fact. The first is a general lack of consensus on the markers that have to be used for studying such cells and/or the use of different protocols in different studies. The precise FCM identification of Tregs is not that simple, likely because of the number of markers that have to be considered. Indeed, Tregs are CD3+,CD4+ cells characterized by the intracellular expression of the forkhead family transcription factor-box P3 (FoxP3) and by high level of the interleukin-2 receptor α-chain (IL-2Rα and CD25). However, both these markers are also transiently overexpressed by effector T lymphocytes during their activation. Moreover, CD4+ FoxP3-negative T cells with regulatory function have been described, and thus FoxP3 cannot be considered the unique Treg-related marker. The importance of interleukin-7 receptor α-chain (IL-7Rα and CD127) for the identification of Treg became evident: T cells with regulatory function show a CD127 low or negative expression. Furthermore, it has been shown that in humans, a subset of FoxP3+ cells, defined as effector/memory-like T cells, can constitutively express the ectoenzyme CD39 ([12]). In any case, the analysis of FoxP3, CD25 high expression, and CD127 remains the most used, even if likely not the better, procedure for CD4+ Treg identification.

The second reason is due to the fact that Tregs likely play different roles in different phases of the infection and that patients in a different stage of the disease can be quite different. Expansion or even a high activity of Tregs could represent a mechanism for the control of the immune hyperactivation typically present during HIV infection. However, Tregs could also inhibit the antiviral immune response, thus weakening HIV-specific responses and favoring the chronicization of the infection. The resulting balance between contrasting outcomes not only on the amount but also on the meaning of Tregs might have critical implications in understanding the pathogenesis of the infection and its immune modulation.

Another population of CD4+ T cells exists, able to produce IL-17, and named Th17 ([13]). These cells produce proinflammatory cytokines such as IL-17 (IL-17A), IL-17F, IL-22, and IL-21 and are regulated by various cytokines (i.e., TGF-β, IL-6, IL-23, and IL-21) and transcription factors such as RORγt, RORα, AhR, and the NF-κB family member IκBζ. Th17 cells play a main role in various inflammatory and autoimmune diseases, including rheumatoid arthritis, psoriasis, systemic lupus erythematosus, and multiple sclerosis, and are also important in mucosal immunology, as they are able to respond to bacterial and fungal antigens. It has been shown that HIV infection causes the disruption of gastrointestinal mucosa, which results in loss of immune cells, translocation of microbes and their products, and chronic immune activation. Thus, changes in Th17 cells could play an important role in HIV pathogenesis, as revealed by the fact that they are preferentially lost from the gastrointestinal tract of HIV+ individuals and ameliorate with HAART ([14]). In addition, in this case, it is notable that since the identification of this subset ([15]), FCM has been largely used to analyze the intracellular presence of IL-17, typically by using a polychromatic approach that combined the detection of two or more cytokines such as IL-4 or IFN-γ.

CD8 T Cells and HIV Infection

  1. Top of page
  2. Abstract
  3. The Epidemics
  4. Changes in CD4+ T Cells, the Main Target of HIV, and in Their Subtypes Have Been Identified by Using FCM
  5. The Importance of T-Cell Activation and CD4+ T-Cell Subtypes
  6. CD8 T Cells and HIV Infection
  7. Natural Killer Cells and HIV Infection
  8. Alterations in the B-Cell Compartment
  9. The Mononuclear Phagocytic System
  10. A Space for FCM Analysis of Soluble Molecules
  11. Conclusions
  12. Literature Cited

Besides the loss of CD4 T cells, several changes in the different T-cell subsets have been described during different phases of HIV infection. In general, CD8+ cytotoxic T lymphocytes (CTLs) control the replication of a huge number of viruses; however, during HIV infection, they are not always able to exert such role. Indeed, FCM has revealed phenotypic and functional changes of CTLs, allowing to highlight several key, general concepts such as not only the already quoted importance of the chronic activation but also the exhaustion and accelerated senescence of T cells ([16, 17]), as well as the increased expression of natural killer (NK) inhibitory receptors in virus-specific CTLs ([18]). Multiple aspects of senescent CTLs are related to the failure of viral control, including the loss of costimulatory molecules, accumulation of terminally differentiated cells, expression of inhibitory receptors, and tendency to undergo apoptosis.

Since the beginning of the epidemics, FCM has permitted to identify the importance of different CD8+ T-cell subsets expressing activation molecules (CD38, HLA-DR, CD45RA, and CD45R0), membrane receptors involved in the costimulation signaling (CD27 and CD28), molecules that regulate T cells [programmed death-1 (PD-1), CD95, and CD178] or are involved in cell trafficking (CD11a and CD62L), cytokine receptors (CD127, CD25, and CCR7), and finally molecules expressed during late differentiation (CD57 and KLRG1). These markers allowed, first of all, to discover the importance of CD8+ T-cell activation: HLA-DR upregulation is a hallmark of HIV disease progression, and high expression of CD38 is associated with a poor prognosis, whereas the percentage of CD8+ T cells expressing HLA-DR in the absence of CD38 correlates with slower progression ([19-21]). Recently, CD8+ T-cell activation was found predictive of the length of the period without treatment in patients undergoing CD4-guided treatment interruption ([10]).

A molecule named PD-1, a member of the CD28 family, provides a negative costimulatory signal to T cells, inhibiting their activation, is involved in apoptosis, and is a crucial negative regulator of T-cell function during HIV infection. PD-1 is highly expressed on HIV-specific CTLs, and CTLs specific for less chronic or acute viruses had lower expression of PD-1 ([22-25]). An inverse relationship between HIV plasma viral load and PD-1 expression on HIV-specific CTLs was reported, and HIV-specific CTLs from patients defined as “long-term nonprogressors” have lower expression of PD-1 than progressors ([26]).

HIV infection affects the balance among different CD8+ T-cell subsets, shifting their phenotype toward a more senescent pattern. CD28 and CD57 are related to T-cell senescence: during chronic infection, CD8+ T cells expressing CD28 are progressively replaced by CD28− cells that exhibit characteristics similar to cells that have repeatedly undergone antigen-driven proliferation ([27, 28]). Similarly, CD57 defines a highly cytotoxic subset of senescent T cells (containing granzyme A and B) that are expanded in HIV infection ([29, 30]).

HIV+ patients also show a generalized deregulation of CD95 (Fas) and CD178 (FasL), which are key molecules in the process of apoptosis ([31-33]). Senescent CTLs also express molecules associated with the CD95/CD178 apoptotic pathway. The accumulation of activated and/or senescent CTLs in HIV+ individuals suggests that the loss of less differentiated cells is a crucial phenomenon and that the cell types that remain are scarcely able to control the infection.

FCM facilitated the comprehension of the perturbation of CTL homeostasis caused by HIV. Based on the combination of a few molecules (CD45 isoforms, CD62L, and CCR7), T lymphocytes (including CD8+ cells) have been first classified in naïve, central memory, effector, and senescent/terminally differentiated cells. Each different type has diverse functional capacities. Naïve and central memory cells expressing CD127 (the α-chain of the receptor for IL-7, a cytokine that is crucial for CTL survival and activity) are progressively replaced with terminally differentiated late-stage cells ([34, 35]). This phenomenon is well evident either during the acute or the chronic phases of the infection. Moreover, during treatment interruptions (where some events occurring during primary infection take place), an expansion of effector and terminally differentiated CD8 T cells expressing CD127 exists; this indicates that a subset of transitional memory CD8 T cells with no complete differentiation to effectors could be induced after the reactivation of the virus ([36]). Indeed, the expression of CD127 on CTLs is significantly decreased in progressive HIV disease, whereas effective HAART results in its recovery ([37]). Observations of impaired IL-7 activity in HIV+ individuals have thus suggested that CD127 plays an important role in the immunopathogenesis of HIV infection and its control. In addition, a soluble form of CD127 (sCD127) is upregulated in the plasma of HIV+ individuals. Thus, CD127 is considered a possible marker of disease prognosis, and related information may contribute to the development of novel cytokine-based therapeutics (reviewed in Ref. [38]).

Further studies took into account not only changes in CTL phenotype but also their function. Using intracellular cytokine staining techniques, the type and quality of HIV-specific CTLs that are actively involved in controlling the virus have been investigated by ascertaining which chemokines and cytokine (i.e., MIP-1β, IFN-γ, IL-2, TNF-α, and CCL3) are secreted on specific stimuli, along with the detection of a degranulation marker like CD107a (Fig. 1). This is a crucial issue, as sometimes a discrepancy can be present between the presence of specific CTLs by and their capability to exert cytotoxicity.

image

Figure 1. Identification of the intracellular presence of cytokines (interleukin-2 and interferon-γ) and of the degranulation marker CD107a in CD4+ and CD8+ T cells from a HIV+ patient after in vitro stimulation with the superantigen SEB. Note the different expression of intracellular IL-2 (highly expressed by CD4+ T cells) or IFN-γ and CD107a (highly present among CD8+ T cells). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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HIV-specific CD8+ T lymphocytes able to exert multiple functions in the same moment have been extensively studied during all phases of HIV infection. The magnitude of the CD8+ T-cell response well correlates with the viral load ([39-41]), and the presence of HIV-specific polyfunctional CD8+ T cells is associated with the control of viral replication. Recent studies have been performed on patients defined as “élite controllers,” who have undetectable viremia and a high number of CD4+ T cells ([42]). They are characterized by a high frequency of HIV-specific polyfunctional CD4+ T cells, along with CD8+ T cells that have multiple functions, including the capacity to secrete IL-2, that proliferate rapidly, and upregulate perforin (reviewed in Ref. [43]). In contrast, HIV-specific CTLs from patients in whom the infection progresses generally secrete fewer cytokines or chemokines (such as IFN-γ or CCL3) and proliferate poorly ([44]). Finally, the recently observed secretion of IL-21 by HIV-specific CD8 T cells from élite controllers has been linked with the maintenance of CD8 T-cell pool and associated to the secretion of IL-2, which is crucial for the control of virus replication ([45]).

Natural Killer Cells and HIV Infection

  1. Top of page
  2. Abstract
  3. The Epidemics
  4. Changes in CD4+ T Cells, the Main Target of HIV, and in Their Subtypes Have Been Identified by Using FCM
  5. The Importance of T-Cell Activation and CD4+ T-Cell Subtypes
  6. CD8 T Cells and HIV Infection
  7. Natural Killer Cells and HIV Infection
  8. Alterations in the B-Cell Compartment
  9. The Mononuclear Phagocytic System
  10. A Space for FCM Analysis of Soluble Molecules
  11. Conclusions
  12. Literature Cited

NK cells represent a highly specialized lymphoid population that exert by a potent cytolytic activity against tumor or virus-infected cells (see the review by Montaldo et al., in this issue). NK cells function is orchestrated by a balance of inhibitory and activating receptors. NK cells inhibitory receptors (KIRs) prevent NK cell-mediated attack of normal HLA class I+ autologous cells, whereas natural cytotoxicity receptors (NCRs) are the activatory receptors such as NKp46, NKp44, and NKp30 ([46]). NK cells have been classically defined by the expression of two cellular markers: CD56, the neural cell adhesion molecule, and CD16, the Fc receptor IIIa. These markers allowed the discrimination of at least three distinct NK cell subsets with different functions. Cytolytic activity is mostly confined to the CD56dim,CD16+ subset, whereas CD56bright,CD16− is responsible for cytokine production ([47]). Monitoring of NK cell effector functions was for a long time limited to the usage of chromium (radioactive or nonradioactive) release assays measuring direct cytotoxicity ([48, 49]). However, FCM assessment of NK cell degranulation by the detection of cell surface CD107a was recently described as a surrogate for measurement of cytolysis ([50]). In particular, the combination of CD107a and intracellular cytokine staining should allow the simultaneous assessment of several distinct functions at the single-cell level ([51, 52]).

Following infection, the distribution of NK cell subsets is altered ([53, 54]), and these cells display a decreased ability to kill virus-infected target cells and interact with other cellular components of the adaptive immune system ([55]). During chronic HIV infection, NK cell cytotoxicity and cytokine secretion are impaired and display a reduced capacity to respond to IFN-α and to produce high amounts of IFN-γ and TNF-α along with low amounts of perforin ([56]). These deficiencies start earlier in the course of disease: the impairments are associated with expansion of an “anergic” NK cell subset expressing CD16 and relatively low levels of CD56 ([57, 58]). Besides, CD56bright CCR7− NK cells are characterized by increased cytolytic potential, higher activation states that strongly correlate with HIV viral load ([59]). In addition, the impaired NK cell cytolytic function in viremic HIV-1 infection was found associated with a reduced surface expression of NCRs (NKp46, NKp30, and NKp44) ([60]), and activated (HLA-DR+CD69+) peripheral NK cells expressed an NCR dull phenotype ([61]).

HAART does not significantly influence the recovery of NK cell function, as IFN-γ production ([62]) and NCR expression may be persistently impaired even after successful therapy and virus control in patients with CD4+ T cell count > 500 μL−1 ([63]). NK cells from HIV patients receiving HAART have reduced expression of key signaling proteins that are required for antibody-dependent cellular cytotoxicity; however, the factors causing this phenomenon are unknown ([64]). Moreover, virologically suppressed HIV patients show activation of NK cells and persistent innate immune activation ([65]).

Few data that evaluate the profile of NK cells during structured therapy interruption (STI) in chronic HIV-1 infection are available. Patients who present the lowest levels of total NK cells and KIR and NKG2A receptor expression after STI showed the poorest virology or immunology outcomes ([66]), whereas decreased proportions of CD56brightCD16+/− NK cells and a reduction of IFN-γ-producing NK cells have been found in patients under CD4-guided treatment interruption ([60]).

A small subset of T lymphocytes, named NKT cells, expresses surface markers of both T and NK cells [including the T-cell receptor (TCR)] and is an important bridge between the innate and the adaptive immune responses. These cells can be activated in antigen-dependent and -independent way and are able to produce both Th1/Th2 cytokines. A subset of NKT cells, named invariant NKT (iNKT) cells, expresses a highly restricted TCR (Vα24-Jα18;Vβ11) and are important in several infectious diseases even if they represent less than 1% of total lymphocytes ([67]). The combination of phenotypic markers together with the improvement of new technologies, such as those based on the acoustic focusing of the cell flux through the laser beam, which allows the acquisition of up to 10 million events in few minutes, is now permitting the quantification and fine analysis of such rare populations. As shown in Figure 2, the identification of iNKT cells can be performed by specific mAbs that recognize the CDR3 region of the invariant TCR or by using α-GalCer-loaded CD1d tetramers. Interestingly, most iNKTs express the NK receptor CD161, but can be CD4+, CD4−CD8−, or CD8+ ([68]). All iNKT cells express high levels of CXCR3 and CXCR4 and display an effector/memory phenotype ([69]). The distinctive feature of iNKT activation is the rapid production of a vast array of cytokines and chemokines during viral infection ([70]).

image

Figure 2. Analysis of the expression of CD161 on iNKT cells expressing CD4 or CD8 molecules from a healthy donor and a patient with HIV infection. Based on physical parameters, a gate was set on lymphocytes (A), then on CD3+ cells (B), and on DUMP− cells (C). iNKT cells were recognized by the positivity to anti-Vα24Jα18Vβ11 mAb (D). Among iNKT cells, the expression of CD4 and CD8 markers was evaluated in a control donor (E) and in a patient with HIV (F). In the healthy control, CD161 was then analyzed on iNKT,CD8+ cells (G), iNKT,CD4−CD8− cells (H), and iNKT,CD4+ cells (I). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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NKT and iNKT cells are depleted in HIV infection, likely because these cells express the viral coreceptor CCR5 and are susceptible to infection ([71-73]). The virus not only causes selective depletion of these cells but also interferes with their activation by antigen-presenting cells (APCs) through downregulating APC expression of CD1d. Moreover, the CD4+ NKT cell subset is rapidly lost in the majority of infected subjects, and the recovery of these cells in response to therapy is typically slow ([74]). The percentage of circulating NKT cell increases significantly within 3 months of initiation of successful therapy, similar to what is observed for the reconstitution of CD4 T cells ([75]). Some patients maintain relative numbers of NKT cells even in chronic HIV-1 infection, but NKT subset displays functional impairment ([76]).

An increased expression of PD-1 on iNKT was reported among HIV-1 patients ([76]), even such levels were not significantly correlated with IFN-γ production or proliferative capacity and PD-1 blockade did not restore iNKT function. In response to α-GalCer stimulation, CD4+/− iNKT cells exhibit reduced proliferation and production of IFN-γ, TNF-α, and IL-4 ([77]). In any case, more data are required to address the dysfunction of these subsets during HIV infection.

Alterations in the B-Cell Compartment

  1. Top of page
  2. Abstract
  3. The Epidemics
  4. Changes in CD4+ T Cells, the Main Target of HIV, and in Their Subtypes Have Been Identified by Using FCM
  5. The Importance of T-Cell Activation and CD4+ T-Cell Subtypes
  6. CD8 T Cells and HIV Infection
  7. Natural Killer Cells and HIV Infection
  8. Alterations in the B-Cell Compartment
  9. The Mononuclear Phagocytic System
  10. A Space for FCM Analysis of Soluble Molecules
  11. Conclusions
  12. Literature Cited

Because CD4+ T cells represent the main target of HIV infection, less attention has been devoted to the analysis of B cells phenotypic and functional alterations in B cells from HIV+ patients. In the early years of HIV epidemics, the efforts were focused on the analysis of abnormalities in the production of immunoglobulins, and then phenotypic and functional characteristics of B cells have been deeply investigated. Technical advancements in FCM have allowed a profound increase in the knowledge of B-cell physiology. Indeed, while the identification of the main T-cell subsets was possible even in the early 1980s, only from the second half of the 1990s, when a precise marker of memory B cells have been identified, the fine analysis of B-cell subsets became possible ([78, 79]).

In healthy human subjects, B cells develop in the bone marrow, then migrate into the periphery, convert into transitional cells (TRs, further divided into TR1, TR2, and TR3 cells), and finally mature in naïve B cells. After contacting the antigen, naïve B cells activate and differentiate into plasma cells, able to secrete specific Abs. When infections end, only a minority of specific B cells survives and constitutes the pool of resting memory B cells ([79]). These B-cell subpopulations are identified in most studies by different expression of CD10, CD19, CD27, CD10, and CD21; other markers such as CD24 or CD38 can be added for a more fine analysis of subsets of TR B cells ([80]). The picture is even more complicated by the presence of B1 cells, whose markers and meaning in peripheral blood is still a matter of debate ([81-85]).

In addition, the first abnormalities in the B-cell compartment during HIV infection were described 30 years ago ([86]), when hypergammaglobulinemia was reported in patients with AIDS. The apparent hyperactivity of B cells was paradoxically associated to a deficient primary response, in terms of defective production of specific antibodies to bacterial antigens ([87]). However, by means of two-color FCM, only in 1987, the first phenotypic alterations in circulating B-cell subsets have been clearly characterized as an increase in the number of activated B cells (identified as transferrin receptor positive cells) and a decrease in resting B cells ([88]).

It was during the late 1990s, after the observation that the expression of CD27 could be used to distinguish naïve from memory B cells ([78]) that an in-depth analysis of B cells phenotype and function has begun. The increase of B-cell activation was further confirmed, an association with the higher expression of CD126 was observed (either on CD19+,CD71+ or CD19+,CD71− cells) ([89]), and a reduction of circulating memory B cells (CD19+,CD27+) ([90]) was evidenced. Then, a seminal paper evidenced the appearance of a CD19+ cell subset characterized by a reduced level of CD21 on the cell surface ([90]); this CD21low subset showed a reduced capacity to proliferate and a higher immunoglobulin secretion rate when compared with the CD21high counterpart ([91]). Further in vitro functional analysis evidenced a reduced expression of CD80/CD86 and CD25 costimulatory molecules by B cells from viremic HIV+ patients after B-cell receptor engagement ([92]).

The factors contributing to the hyperactivation of B cells observed in HIV patients, however, are still not completely clear. Several studies showed an impairment in the plasma levels of proinflammatory and/or homeostatic cytokines, including TNF-α ([93]), IL-6 ([94]), IL-7 ([95]), and BAFF ([96]), and it was hypothesized that this could indirectly affect B cells activity.

The identification of transitional, naïve, and resting memory B cells, along with the analysis of activation markers ([97, 98]), has given additional information. Concerning resting memory B cells, their frequency was strongly reduced in HIV+ individuals; this reduction was paralleled by increased levels of naive cells, activated mature B cells (CD20+,CD21low,CD27+), and plasma cells (CD20−,CD21low,CD27+) ([99-101]). The expansion of an “abnormal” B-cell subset in the peripheral blood of HIV+ subjects emerged. In particular, very high levels of immature/transitional B cells, as defined by the expression of CD10 surface marker, were observed. These cells showed a phenotype that resembled those of TR1 (CD27−,CD10high/CD21low) or TR2 (CD27−,CD10int,CD21high), were less responsive to BCR engagement, were characterized by higher tendency to undergo apoptosis, and appeared to be correlated with disease progression and IL-7 levels ([95]). As IL-7, necessary for B-cell development in the bone marrow, is augmented in HIV+ patients as an attempt to compensate CD4+ T cell loss, the presence of CD10+ immature/transitional B cells in the blood could likely be considered as an indirect consequence of the decline of CD4+ T cells. Later, FCM and in vitro functional studies showed the expansion of a subset of memory B cells with a tissue-like, “exhausted” phenotype in HIV+ patients. These cells were defined as CD19+,CD20high,CD21low,CD27− and were characterized by a pattern of homing and inhibitory receptors different from classical memory cells. In particular, they expressed the inhibitory molecule FCRL4 and showed a very poor capacity to expand in response to specific antigen ([102]). The percentage of circulating tissue-like memory B cells was correlated with viremia and a reduced immunosurveillance ([103]).

The advent of HAART in 1996 had a deep impact on B-cell compartment as well. Potent therapy normalizes B-cell count and the relative percentages of the main B-lymphocyte subsets (including immature/transitional and memory/activated cells) ([104]). Moreover, therapy can normalize CD70, CD71, CD80, and CD86 expression ([92, 105]). However, even potent treatments are not able to fully restore memory B-cell depletion during chronic infection at the level observed in healthy individuals ([104, 106, 107]).

The Mononuclear Phagocytic System

  1. Top of page
  2. Abstract
  3. The Epidemics
  4. Changes in CD4+ T Cells, the Main Target of HIV, and in Their Subtypes Have Been Identified by Using FCM
  5. The Importance of T-Cell Activation and CD4+ T-Cell Subtypes
  6. CD8 T Cells and HIV Infection
  7. Natural Killer Cells and HIV Infection
  8. Alterations in the B-Cell Compartment
  9. The Mononuclear Phagocytic System
  10. A Space for FCM Analysis of Soluble Molecules
  11. Conclusions
  12. Literature Cited

Monocytes, macrophages, and dendritic cells (DCs) are very heterogeneous cell types, but can be easily investigated by FCM ([108]). Within the mononuclear phagocytic pool, monocytes/macrophages are often distinguished from DC by differential expression of surface makers such as human epidermal growth factor module-containing mucin-like receptor 1 (EMR1), CD11b and CD18 (also known as MAC1), CD68, and Fc receptors. However, few, if any, combinations of known markers can definitively distinguish macrophages from myeloid DC (MDC), mainly because these populations exist on a continuum of development from common myeloid progenitors ([109]).

Concerning human monocytes, three subsets can be identified according to the expression of CD14 and CD16 ([110]): CD14high,CD16− “classical” monocytes, CD14high,CD16+ “intermediate” monocytes, and CD14dim,CD16+ “nonclassical” monocytes (Fig. 3). Classical monocytes secrete proinflammatory cytokines, exert phagocytosis, and produce reactive oxygen species ([111, 112]). Intermediate monocytes express very high levels of major histocompatibility complex (MHC) class II molecules and accessory molecules, exhibit high levels of phagocytosis, and produce the highest amounts of both proinflammatory and anti-inflammatory cytokines in response to Toll-like receptor (TLR)-4 agonist stimulation ([113]). Nonclassical monocytes respond to viruses and nucleic acids with production of proinflammatory cytokines via TLR-8 and TLR-9 pathways ([113]). Additional surface molecules can be used to further identify other monocyte subsets. CD14high,CD16− monocytes express the CC-chemokine receptor (CCR)−2 and CD11b (MAC-1) at high levels, whereas CD14dim,CD16+ cells express high levels of the CX3C chemokine receptor for fractalkine (CX3CR1) and CD11a (LFA-1). Finally, CD14high,CD16+ monocytes express high levels of CCR5 and CCR2 (reviewed in Ref. [111]).

image

Figure 3. Identification of different monocyte subpopulation in a patient with chronic HIV infection, and expression of TREM and VEGFR1 in all these cells. A gate was set on cells that were CD14+, CD16+, or double positive (A). Then, a gate was set to remove CD16+,HLA−DR− cells (i.e., NK cells) (B), and the expression of CD14 and CD16 was reanalyzed (C) to distinguish classical (CL), nonclassical (Non-CL), and intermediate (Int) monocytes (according to Ref. [110]). Finally, the presence of TREM (D) or VEGF-R1 (E) was detected in classical monocytes. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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HIV infection is characterized by a persistent systemic immune activation and chronic inflammation that can be responsible for a variety of side effects other than immunological diseases, including diseases characterized by chronic inflammation. Previously, it was suggested that alterations of the mucosal immune system due to CD4+ T cell loss in the lamina propria cause the translocation of microbial products from the gastrointestinal tract to portal and systemic circulation, causing and sustaining the persistent immune activation ([114]). Microbial products elicit potent proinflammatory responses by activating a number of innate receptors: nucleotide-binding oligomerization domain 1 (NOD1) and NOD2, as well as TLR-2, TLR-4, TLR-5, TLR-6, and TLR-9, which are expressed by many cell types. Monocytes, macrophages, and DCs binding of these receptors to microbial products activates a signaling cascade within the cell, leading to the production of the proinflammatory cytokines IL-1β, IL-6, TNF-α, and Type I interferons (IFNs), including IFN-α and IFN-β. Although these responses may be beneficial, if not essential, to the host in the setting of acute infections, they may contribute to disease pathology in numerous situations, including chronic HIV infection.

Monocytes are relatively resistant to HIV infection, whereas differentiated macrophages are highly susceptible ([115]); CD14+,CD16+ monocytes are preferentially infected by HIV ([116]). Monocytes from HIV+ patients possess many characteristics of activated cells, such as spontaneous production of proinflammatory cytokines, expression of CD38, CD69, CD11b, HLA-DR, and CD86, and decreased CD62L (reviewed in Ref. [117]). Early studies indicated that CD14+,CD16+ monocytes increase in HIV+ individuals and are expanded in those who do not take therapy or discontinue treatment. On the contrary, in patients under effective HAART, the expression of CD16 is similar to that of uninfected controls ([118]). In HIV+ patients, CD69 and HLA-DR expression well correlates with plasma levels of lipopolysaccharide, which is an indicator of microbial translocation ([119]).

Recently, the cytometric analysis of CD62L, CD11b, and CD115 has revealed that in HIV+ young men, phenotypic and functional changes in monocytes exist that are similar to those observed in elderly uninfected individuals. This suggests that HIV may accelerate age-related changes in monocytes and that these defects persist even in virologically suppressed HIV+ individuals ([120]).

Nonclassical and intermediate monocytes are increased during the infection and express high levels of tissue factor and CD62P that are correlated are related to viremia, to T cell activation, and to plasma levels of IL-6. Interestingly, the profile of monocyte activation in uncontrolled HIV infection mirrors that reported during acute coronary syndromes in uninfected persons. Thus, HIV-related immune activation and inflammation could contribute to the development of a proatherosclerotic state ([121]). Clinical and epidemiological studies have indeed consistently connected HIV infection with increased risk of cardiovascular diseases, and this field is now under accurate investigations.

DCs represent a very heterogeneous population of specialized migratory APCs, ubiquitously distributed both in lymphoid and nonlymphoid tissues, which can induce either immunity or tolerance. Under inflammatory conditions, blood monocytes can develop many of the phenotypic features and functions of DCs ([108]). Three major DC subsets have been described, that is, MDC, plasmacytoid DC (PDC), and Langerhans cells, which differ in ontogeny, phenotype, and function. PDCs are involved in antiviral immunity, are capable of sensing viral single-stranded RNA through TLR-7 and bacterial CpG DNA through TLR-9, and predominantly produce Type I IFNs. Conversely, MDCs sense both bacterial and viral pattern motifs through a broader range of TLRs and are involved in the induction of Th1- and Th2-type responses through the production of cytokines such as IL-12 and IL-10. Both subsets exhibit a functional plasticity in directing T-cell responses ([122]).

MDC and PDC can be defined by the expression of specific cell surface markers and TLRs. MDCs express BDCA-1 (CD1c) and CD11c, whereas PDCs express BDCA-2 (CD303), BDCA-4 (CD304), and CD123. TLR3 is selectively expressed in MDCs, whereas PDCs express TLR7 and TLR9 ([123-125]). Productive viral replication occurs in human monocyte-derived DCs for up to 45 days, and these cells may survive longer within the lymph nodes due to cytokine stimulation in the microenvironment, which may help to spread HIV-1 infection and to maintain viral reservoirs ([126]).

Alterations in DC numbers, phenotype, and function exist in HIV+ subjects ([127-131]). Peripheral MDC and PDC numbers are decreased during acute HIV infection ([132]). Both cellular redistribution to lymph nodes and cell death may contribute to lower the amount of peripheral blood DCs ([133, 134]). Blood MDC from untreated HIV-1-infected subjects displays a proapoptotic profile that can be partially reversed with viral suppression, suggesting that the death of DC may be a factor contributing to their depletion during chronic, untreated HIV disease ([135]).

Costimulatory or activation markers (such as CD40, CD80, CD83, CD86, CD38, HLA-DR, and PD-L) of both MDC and PDC have also been investigated in HIV+ subjects: MDC and PDC display an increase in the activation phenotype, and the levels of PD-L1+ MDC was related to viremia ([136]). On the contrary, PDCs express moderately higher levels of PD-L1 and lower levels of PD-L2 in untreated HIV+ patients when compared with controls or with HAART-treated patients, whereas no significant changes were observed in MDCs.

A Space for FCM Analysis of Soluble Molecules

  1. Top of page
  2. Abstract
  3. The Epidemics
  4. Changes in CD4+ T Cells, the Main Target of HIV, and in Their Subtypes Have Been Identified by Using FCM
  5. The Importance of T-Cell Activation and CD4+ T-Cell Subtypes
  6. CD8 T Cells and HIV Infection
  7. Natural Killer Cells and HIV Infection
  8. Alterations in the B-Cell Compartment
  9. The Mononuclear Phagocytic System
  10. A Space for FCM Analysis of Soluble Molecules
  11. Conclusions
  12. Literature Cited

In the past, FCM has also given a substantial contribution to the detection of soluble molecules such as cytokines or chemokines, which are of major interest in monitoring HIV infection and its treatment ([137]). Indeed, the development of sophisticated technologies that allow the simultaneous measurement of cytokines and chemokines derives from the need to deeply characterize the magnitude and several functional aspects of the immune response, which follows the activation of distinct pathways and can be expressed in different moments. Immune functions include a large variety of features, such as proliferation, stimulation, cytotoxicity, chemotactic responses, regulation, inflammation, and Th1/Th2/Th17 plasticity, and the discovery of new functions always requires further information required for a better understanding and characterization of such functions.

Traditionally, secreted cytokines and chemokines were measured by enzyme-linked immunosorbent assay (ELISA), which use immobilized antibody to capture a soluble ligand detected by a second reporter antibody. However, ELISA is not suited for high-throughput multiplex analyses, and, at least in immunology, has been joined by cytokine bead array assays. These tests use FCM to simultaneously detect and quantify multiple proteins, as cytokines are captured by beads with a unique fluorescent intensity so that beads can be mixed and run together in a single tube ([138]). Cytokine bead array assay can thus be used to detect cytokines both in plasma and in supernatants of PBMC or any other cell source. The same approach can be used for detecting those molecules still present on the plasma membrane. In the field of HIV research, all of these assays have been largely used.

Recently, a multivariate “cytokinomics” approach, which combines cytometric bead array technology, FCM, and multivariate analysis, has been performed to investigate Th1, Th2, and Th17 cytokine profile of HIV-infected patients ([139]). The simultaneous analysis of plasmatic levels of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A revealed that minor changes are present in HIV patients during the asymptomatic phase of the infection, but such changes increase during the progression of the disease ([140]). Cytokine bead array assay has also been used to assess 12 cytokines and chemokines in supernatants collected from stimulated PBMC from individuals enrolled in the HVTN 068 candidate HIV-1 vaccine clinical trial in order to analyze the quality and quantity of the immune response ([141]). Then, plasma concentrations of 22 cytokines and chemokines have been evaluated by multiplex bead approach to measure baseline levels of T-cell activation and regulatory T cells, HIV-specific T-cell cytokines, and proliferation responses ([142]). This study revealed that élite controllers and the so-called viremic controllers are characterized by high levels of macrophage inflammatory protein 1α and low levels of monocyte chemotactic protein 1 and TGF-β.

The quantification of plasma levels of cytokines and chemokines has also been used as a marker for the response to therapy or for investigating other clinical situations. For example, the Strategies for Management of Antiretroviral Therapy (SMART) trial studied patients who interrupted treatment in comparison with those who maintained their successful therapeutic regimens. The arm where patients stopped therapy showed significant increases of the proinflammatory molecules TNF-αIL-10 and CXCL10 when compared with the arm where patients continued therapy ([143]). This showed the importance of viral rebound in triggering a generalized inflammatory reaction.

The expression of cytokines can also be measured by intracellular labeling with fluorochrome-conjugated anti-cytokine mAbs (polyfunctionality). In this case, FCM gives the possibility to investigate the presence of different molecules in the same cell ([10, 36]).

Another tool should be mentioned that links FCM to soluble molecules. This tool relies on the use of multimerized peptide–MHC to make soluble peptide–MHC-soluble tetramers, which identifies antigen-specific T cells, without considering their function. Indeed, such tetramers allow the quantification and characterization of circulating antigen-specific T cells ([144]). With the limitation of the need to use MHC–tetrameric complexes with the same HLA specificity as that of the subject under analysis, tetramers can be used in a relatively simple way to visualize antigen-specific T cells by FCM ([145]) and also to isolate peptide-specific T cells, in a manner that requires minimal in vitro manipulation ([146-148]). In the context of HIV, this approach has been used by several groups to demonstrate that (i) HIV-specific CTLs have a role in the control of HIV infection ([145]); (ii) CCR5 is functionally expressed on antigen-specific memory and effector CD8+ T cells and has a critical role in the migration of these cells to inflammatory tissues and secondary lymphoid tissues ([149]); (iii) replicating viral populations are required to maintain high frequencies of HIV-1 epitope-specific CD8+ T cells (in particular Gag-specific CD8+ T cells) in asymptomatic chronically infected patients ([150]); and (iv) a significant proportion of HIV-specific CD8 T cells are functionally unresponsive in vivo in chronic HIV infection ([151]).

The efforts to develop and optimize new technologies that allow the systematic analysis of cytokine and chemokine network derives from the observation that these molecules are important players in the immunopathogenesis of HIV, and a comprehensive view of the mechanisms that govern their homeostasis has the potential to provide insights for the identification of novel therapeutic targets.

Conclusions

  1. Top of page
  2. Abstract
  3. The Epidemics
  4. Changes in CD4+ T Cells, the Main Target of HIV, and in Their Subtypes Have Been Identified by Using FCM
  5. The Importance of T-Cell Activation and CD4+ T-Cell Subtypes
  6. CD8 T Cells and HIV Infection
  7. Natural Killer Cells and HIV Infection
  8. Alterations in the B-Cell Compartment
  9. The Mononuclear Phagocytic System
  10. A Space for FCM Analysis of Soluble Molecules
  11. Conclusions
  12. Literature Cited

FCM has always been playing a crucial role not only in HIV research but also in monitoring the treatment of this infection. Improvements in all aspects of FCM gave, and are giving, substantial contributions to the fight against the infection.

At present, in our opinion, there are two areas where more efforts are needed. On one side, we need sophisticated instruments that allow the identification of unprecedented numbers of parameters and functions in the same cell. They will facilitate our understanding of the strategies that HIV uses to evade the immune response. However, hyperparametric approaches have extremely high costs that are difficult to afford, especially when research is suffering from the global economic crisis.

The second area regards the importance to use high-quality instruments and reagents, first of all monoclonal antibodies, which have to be produced at very low cost, with an absolute reliability. Likely, these tools will be used not only in poor resource settings but also in well-established laboratories, where they could reduce the costs for routine assays. Our community has to thus sustain both these efforts.

Literature Cited

  1. Top of page
  2. Abstract
  3. The Epidemics
  4. Changes in CD4+ T Cells, the Main Target of HIV, and in Their Subtypes Have Been Identified by Using FCM
  5. The Importance of T-Cell Activation and CD4+ T-Cell Subtypes
  6. CD8 T Cells and HIV Infection
  7. Natural Killer Cells and HIV Infection
  8. Alterations in the B-Cell Compartment
  9. The Mononuclear Phagocytic System
  10. A Space for FCM Analysis of Soluble Molecules
  11. Conclusions
  12. Literature Cited
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