B cell immunopathology during HIV-1 infection: Lessons to learn for HIV-1 vaccine design

Vaccine (2008) 26, 3016—3025

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B cell immunopathology during HIV-1 infection: Lessons to learn for HIV-1 vaccine design Alberto Cagigi a, Anna Nilsson a,b, Angelo De Milito c, Francesca Chiodi a,∗ a

Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Nobels v¨ ag 16, S-17177 Stockholm, Sweden Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden c Department of Drug Research and Evaluation, Pharmacogenetic, Drug Resistance, and Experimental Therapeutic Section, Istituto Superiore di Sanit` a, Rome, Italy b

Received 30 October 2007; received in revised form 20 November 2007; accepted 23 November 2007 Available online 17 December 2007

KEYWORDS B cells; HIV-1 vaccination; Chemokine receptors

Summary Induction of broad HIV-1 neutralizing antibodies should be a major goal of an effective HIV-1 vaccine. However, B cells are severely damaged during HIV-1 infection with loss of memory B cells and decline of serological memory. The molecular events leading to B cell damage must be further characterized with the aim of selecting vaccine components allowing preservation of B cell functions. This review focuses on B cell damage and antibody responses in HIV-1-infected patients during vaccination studies with viral and bacterial antigens. In addition novel data indicate that B cell activation may be at the basis of impaired immune responses. © 2007 Elsevier Ltd. All rights reserved.

Introduction B cell responses are important to consider in the context of HIV-1 vaccines, as it has been shown that passive immunization can provide sterilizing immunity against mucosal HIV-1 challenge [1]. Ruprecht and collaborators used the simian-human immunodeficiency virus (SIV) monkey model to show that protection against oral challenge can be obtained by intra-muscular post-exposure prophylaxis with three anti-HIV-1 human neutralizing monoclonal antibodies (Abs) possessing potent cross-clade neutralization activity [1]. In addition, a study from Trkola et al. [2] indicated that passive transfer of human neutralizing Abs can be used to

∗

Corresponding author. Tel.: +46 8 52486315. E-mail address: [email protected] (F. Chiodi).

delay HIV-1 rebound after cessation of antiretroviral therapy. These two studies provided a proof of concept of the relevance of B cell responses during HIV-1 infection, leading to a renewed focus on neutralizing Abs as a correlate of protection for HIV-1 vaccine and during natural infection. That neutralizing Abs may be able to reduce virus yield by directly targeting HIV-1-infected cells as well as free virus has been recently suggested by Hessell et al. showing that removal of the Fc portion of a broadly neutralizing Ab lead to loss of Ab protective activity [3]. This new study opens a new angle of interpretation and investigation in engineering neutralizing Abs with the correct Fc portion able to induce neutralizing activity [3]. In line with these findings, it is essential that a candidate HIV-1 vaccine should be able to induce functional and broadly neutralizing anti-HIV-1 Abs. One important aspect in this context is the selection and presentation of antigens (Ags) to be included in a HIV-1 vaccine. In general the HIV-1

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B cell dysfunctions and HIV-1 vaccine design envelope protein gp120 remains an important target for eliciting neutralizing activity and cumulative work from many investigators suggest that V3 may be an important epitope to target for the induction of protective Abs [4]. Interestingly, it has been shown that the neutralizing capacity present in the sera of three asymptomatic individuals exhibiting broad neutralization can result from more than one specificity, including Abs recognizing the CD4 binding site [5]. Within the CD4 binding site an important epitope for neutralization of HIV-1 has been identified, corresponding to the functionally conserved region that allows for initial attachment of the HIV-1 gp120 to the target cells. In order to maximize the accessibility of this site, variants of the gp120 stabilized in the CD4 bound state have been created [6] and this work may become pivotal to indicate how to access a site which in vivo is probably subjected to multiple strategies of humoral evasion, including conformational masking and self-masquerade glycan.

HIV-1 induces damage to memory B cells and loss of specific Abs The realistic goal of HIV-1 vaccines may not be to prevent infection, but to control HIV-1 associated diseases. For this purpose, following HIV-1 vaccination the preservation of immune functions upon encounter with HIV-1 is pivotal. One important component of HIV-1 pathogenesis is the damage occurring to B lymphocytes [7—9] which are the cells devoted to production of Abs, including anti-viral neutralizing Abs. The B cell damage can be measured by the progressive loss of memory B cells and serological memory to Ags met through vaccination or natural infections, including HIV-1 [8]. The molecular pathway and events leading to loss of memory B cells and serological memory during HIV-1 infection are poorly understood. If a vaccine does not evoke immunity to the HIV-1 components involved in the mechanism of B cell damage, the possibility remains that vaccinated individuals may not be protected from HIV-1 induced B cell damage, thus impairing production of effective anti-HIV-1 neutralizing Abs. Therefore, in addition to improve knowledge on which HIV-1 proteins to include in an effective HIV-1 vaccine and how to present them for the immune system, the damage to the B cell compartment must be taken into account for an effective HIV-1 vaccine. Indeed, the finding that HIV-1 alters the immunology and biology of B lymphocytes was an early observation during the HIV-1 epidemic; the appearance of such disturbances in patients with asymptomatic infection occurred before CD4+ T cell decline [10—11]. In a study including patients with primary HIV-1 infection (PHI) we found a severe and irreversible damage to different B cells populations [12] that are prone to apoptosis and display an abnormal pattern of activation/differentiation markers [12]. Phenotypic and functional alterations on B lymphocytes are often observed in chronically infected patients. Memory is a hallmark of immunity and production of Abs can last for a lifetime. The mechanisms leading to long-time serological memory are poorly understood and three major hypotheses have been presented over the past decade. Zinkernagel and colleagues [13] suggested that long-term Ab memory is driven by a constant differentiation of memory B cells to plasma cells in the presence of Ags.

3017 However, this hypothesis would require persistence of all infectious agents met during a lifetime. Ahmed and colleagues [14] have, on the other hand, shown that mouse plasma cells can be long-lived and are able to produce Abs for several months in the absence of memory B cells or Ags. This hypothesis implies that the same plasma cell must survive for the whole life of an individual and it is debated on whether these cells can have a life span of several decades. However, this must be possible since it has been shown that the Ab levels to smallpox, a virus no longer in circulation, are maintained in humans immunized 25—75 years ago [15]. Lanzavecchia and colleagues presented an alternative mechanism for maintenance of serological memory when showing that human memory B cells proliferate and differentiate into plasma cells in response to polyclonal, non-cognate stimuli [16—17]. The polyclonal stimuli that can activate memory B cells are of two origins; those which activate B cells via Toll-like receptor (TLR)-4 or TLR-9 (such as microbial products and single-stranded DNA motifs) or activated T cells stimulating the B cells via CD40L and cytokines without the presence of Ag-activation. Our group and Nagase et al. were the first to report that HIV-1-infection is characterized by reduced levels of circulating memory B cells [7,18]; in fact memory B cells represent a marker of disease progression because of their correlation to CD4+ T cell counts in chronically infected subjects [19-20]. In line with this, we also found that Agspecific humoral response and total counts of memory B cells are better preserved in long-term non-progressors while the damage to this compartment is already detectable in patients with primary infection [12,19]. Moreover, several studies have reported the loss of both IgM+ and switched memory B cell populations suggesting that loss of memory B cells involves both T-dependent and T-independent humoral immune responses [12,19,21]. The severe alteration to memory B cells is likely a major mechanism underlying the low levels of specific Abs to naturally acquired infections occurring in HIV-1infected patients. In fact, Abs against several opportunistic pathogens (Streptococcus pneumoniae, Salmonella typhi, Pneumocystis carinii and Cryptococcus neoformans) are significantly reduced in HIV-1-infected adults [22—25] and reduction of Abs to naturally acquired infections like measles, varicella and cytomegalovirus is also reported in both infected adults and children [8,26]. The defect of ‘‘serological’’ memory in infected adults may not only affect their own susceptibility to infections [8,27—29], but in the case of HIV-1 positive mothers may severely lower the protection of the newborn against life-threatening infections. Indeed, infants of HIV-1-infected mothers have an increased risk of acquiring measles infection early in life [30—34]. It is highly conceivable that while the defective na¨ıve B cells may be hampered in raising an optimal response to HIV1 Ags and vaccination Ags [35—36], the severe damage to the memory B cells may negatively affect the maintaining of protective Ab levels to both previously and newly encountered pathogens [8,20,29]. The compartment of memory B lymphocytes is an essential component of the immune response to (re-) infections and therefore its function is crucial for the maintenance of serologic memory [16,37]. The complex interplay between decline of B cell memory and the production of ‘‘good-neutralizing Abs’’ during

3018 HIV-1 infection is poorly studied. In HIV-1-infected individuals anti-HIV-1 Abs are detected within weeks from initial infection [38], but on the other hand, neutralizing Abs against anti-HIV-1 appear usually several months after HIV-1 infection [39—42]. The neutralizing activity of sera from HIV-1-infected individuals varies, although it has been shown that broad cross-neutralizing activity in serum is associated with slow progression in SIVinfected macaques [40] and that among HIV-1-infected individuals, rapid progressors have low titers of antiHIV-1 neutralizing Abs following the decline in CD4+ T cells and the consequent loss of T helper activity [43]. As a matter of fact, unclear is also the mechanism leading to decline of neutralizing Abs during the course of HIV-1 infection. Intra-patient, HIV-1 genetic variability has been proposed to be at the basis for loss of anti-HIV-1 neutralizing activity following progression of HIV-1 associated clinical conditions in infected individuals. However, since loss of the cell population devoted to the production of high affinity Abs, e.g. memory B cells, occurs during HIV-1 infection we propose that loss of memory B cells may have a direct impact and role on the poor production of neutralizing Abs occurring during HIV-1 infection. The absence of broadly reactive neutralizing Abs in most HIV-1-infected individuals is likely to be the result of the inability of the humoral immune system to mount adequate B cell responses. Following the introduction of highly active antiretroviral drug (HAART), it has been observed that both HIV-1 specific and HIV-1 non-specific B cell responses decrease after virologically successful therapy [19,44—46], suggesting that low level viral replication is not sufficient to maintain HIV-1 specific Abs and memory B cells. However, an interesting and unexpected finding is that despite the recovery of CD4+ T cell count following HAART, a high proportion of HIV-1-infected adults and children are still unable to maintain protective levels of Abs against vaccination Ags. In fact, although some reports have shown an improvement of humoral immunity against CMV and influenza in short-term treated patients [47—49], studies performed on both adults and children followed for longer periods have indicated the inefficacy of HAART to maintain humoral immunity to Ags like measles, tetanus, influenza and pneumococcus [19,26,50]. Susceptibility to opportunistic infections in HIV-1-infected patients with increased CD4+ T cell counts on HAART remains relevant [51]. Moreover, defects in B cell memory are associated with impaired humoral immune responses in patients on HAART and may be a contributory factor to the increased risk of invasive pneumococcal infection [20—21]. Although most of these studies have been performed on chronically infected patients, we found that HAART on primary-infected patients followed for 6 months did not improve anti-measles Ab levels and induced a reduction of anti-HIV Abs [12].

The impact of CD4+ T and memory B cell counts on vaccination with T cell dependent and independent Ags in HIV-1-infected subjects People infected with HIV-1 are at risk for a variety of infections and therefore immunization against several viral and

A. Cagigi et al. bacterial diseases like pneumococcus, influenza virus, hepatitis B virus (HBV), and Haemophilus influenzae type-B (Hib) are recommended for HIV-infected patients. However, already at an early stage of the epidemic it became evident that HIV-1-infected individuals respond poorly to immunization with common Ags such as pneumococcus or influenza and that the level and duration of the humoral immune responses to vaccination Ags both in adults and children are correlated to the baseline immunodeficiency status and specifically to CD4+ T cell counts in the pre-HAART era [29,52—55]. This has been shown both for T cell dependent Ags (viral Ags) as well as for T-independent Ags (bacterial Ags). In the following section, immune responses in HIV-1infected patients upon vaccination with influenza (T cell dependent Ag) and pneumococcus (T-independent Ag) will be discussed as model Ags. Influenza, a T cell dependent Ag, with its yearly epidemics has been extensively studied and represents a clinical relevant disease to prevent in immunocompromised hosts [56]. Fuller and collaborators examined Ab response and immune activation in HIV-1-infected individuals after immunization with a trivalent influenza vaccine [57]. Only 13 of 29 individuals (45%) responded with a four-fold Ab increase in titers against at least one Ag, most of them within 2 weeks after immunization. The Ab response correlated to CD4+ T cell count and by multivariate analysis, CD4+ T cell count <100 × 106 cells/l was the strongest predictor of a poor Ab response. These data were later confirmed in a prospective vaccination study against influenza in a Dutch cohort of HIV-infected individuals followed for a 3-year period [58]. In this study, post-vaccination Ab titers were significantly lower in HIV-1infected individuals compared to healthy controls and this difference was most striking in patients with a CD4+ T cell count <300 × 106 cells/l and in particular in patients with a CD4+ T cell count <100 × 106 cells/l. As a consequence, the proportion of individuals with a protective Ab titer after vaccination was less in HIV-1-infected individuals with a low CD4+ T cell count. Vaccine boosters were given at regular intervals for 3 years and, interestingly, the pre-vaccination titers found at the 2nd and 3rd years in healthy individuals were higher as compared to the pre-vaccination titers the 1st year indicating that a solid, long-term serological memory was established. In HIV-1-infected individuals with a CD4+ T cell count >300 × 106 cells/l, the pre-vaccination titers on the 2nd and 3rd year tended to be higher than pre-vaccination titers as in controls, whereas the titers were consistently lower in patients with low CD4+ T cell counts. In a recent study [29], the immune response after influenza vaccination was studied in vivo and in vitro. By collecting PBMC at day 7 after immunization, the generation of specific Ab-producing cells by Elispot was studied and no differences were found in the number of Ag-specific Abproducing cells between the infected and control groups. However, in the HV-1 patients the number of influenzaspecific cells correlated to CD4+ T cell counts. At day 54 post-immunization, influenza-specific memory B cells were again measured by Elispot after a 5-day stimulation in vitro. At this time point, a significant higher number of specific memory B cells could be detected in healthy individuals as compared to HIV-1-infected individuals. These findings may

B cell dysfunctions and HIV-1 vaccine design indicate that HIV-1-infected patients are able to respond to immunization but that further differentiation or survival of memory B cells is impaired during HIV-1 infection. Data in support of this hypothesis was reported in a study on primary measles immunization in children with HIV-1, where 88% of infected children sero-converted compared to 93% in un-infected children. The major difference was found in the maintenance of protective titers where the HIV-1-infected children showed rapid waning of immunity compared to uninfected children [59]. Few studies have specifically studied the impact of HAART on immunization responses. In one study [47], the immune response after influenza vaccination in HAARTtreated patients was compared to untreated historical controls and it was found that an increased proportion of patients on HAART responded with a four-fold increase in post-vaccination titers (55% vs. 28%). However, since the patients on HAART had significantly higher CD4+ T cell counts than the historical controls at vaccination it was difficult to draw any conclusion on the effect of HAART per se on immune response. In a more recent study, the immune response to tetanus (recall Ag) and the key limpet hemagglutin (KLH) protein (de novo Ag) were assessed in patients who all received HAART and controls [60]. Interestingly, the CD4+ T cell counts did not differ between patients and controls at the time of immunization but despite this fact, controls responded significantly better with higher titers against both T-dependent Ags. The pre-treatment nadir CD4+ T cell count could predict immune response and in addition, the expression of the costimulatory molecule CD28 on CD4+ T cell

Figure 1

3019 was an independent predictor of response to immunization [60]. To summarize, several studies indicate a crucial role for CD4+ T cell counts when predicting the immunization response against a T cell dependent Ag. The impact of HAART on these immune responses is mainly mediated by an increase in the number of CD4+ T cells during treatment. An initial Ab response to vaccination Ags is obtained in HIV-1-infected individuals followed by poor maintenance of Ab titers, likely reflecting damage to memory B cells. The B cell dysfunctions that may lead to impaired immune response following vaccination with T cell dependent Ags in HIV-1-infected patients are summarized in Fig. 1. Ab formation against several bacterial Ags (mainly capsular polysaccharides) is thought to be a T cell independent process [61]. Bacterial capsular Ags are poorly immunogenic and in general elicit poor memory responses and therefore some bacterial vaccines are conjugated with toxins to provide increased T cell help and improved immune responses. Invasive pneumococcal disease (IPD) is an important cause of morbidity in HIV-1-infected individuals even in the era of HAART, and immunization to protect against IPD could be used as prophylaxis in general clinical praxis. Immune responses against pneumococcus polysaccharide vaccine (PPV) have therefore been extensively studied in HIV-1 patients. Several studies have shown that HIV-1-infected individuals are less likely to respond to PPV than healthy controls, but in those who responded similar IgG levels could be achieved [22,55,62—63]. The Ab response to specific pneumococcus serotypes was followed for 5 years after PPV [22].

B cell defects possibly leading to poor Ab response upon vaccination with T cell dependent Ags during HIV-1 infection.

3020 In this study the mean Ab concentration for serotypes 18C, 19F and 23F was significantly lower in HIV-1 patients with a CD4+ T cell count <200 × 106 cells/l than in healthy controls; on the contrary the mean Ab concentrations, for serotypes 1, 4, 9V, 6B and 14 was similar in all patients and controls. The rate of decline of Ab titers showed no differences between HIV-1-infected individuals and controls over the 5year study. The conclusions drawn from these studies may be that some of the pneumococcus Ags are T cell independent type 2 Ags requiring T cell help for proper induction of immune responses. To further improve the immunization response towards PPV, randomised trails have compared the immune response between conventional 23-polyvalent PPVs and conjugate vaccines against a number of serotypes [64—65]. Ahmed and collaborators concluded that in un-infected individuals conjugate vaccines resulted in higher Ab titers than PPVs, whereas in HIV-1-infected individuals there were no differences in immune response comparing the two vaccine types [64]. In a more recent study, immunization with a conjugate-vaccine followed by PPV vaccination was studied. The Ab response against conjugate-vaccine was lower in patients with a CD4+ T cell count <200 × 106 cells/l than in patients with >200 × 106 cells/l and healthy controls. However, higher Ab concentration was achieved in HIV-1 patients and controls after sequential immunization with conjugatevaccine and PPV than after conventional PPV alone; the mechanisms that underline this observation are yet not clarified. The immunological mechanisms behind the lower Ab response to PPV in HIV-1 patients have been shown to be associated with the clinical status and CD4+ T cell count, but also with B cell defects [19,66]. Abs to PPV are mainly generated from the variable region gene family 3 (VH 3) immunoglobulin family and during HIV-1 infection VH 3 gene expression is dysregulated and B cells expressing VH 3 are deleted. The effect of HAART on the ability to restore Ab production from VH 3 B cells was assessed and only patients treated with HAART could mount PPV specific Abs [66]. Vaccination response after PPV has also been assessed in relation to the composition of the B-cell compartment in infected and un-infected individuals [21] and it was shown that poor responders in the HIV-1-infected cohort had a reduced number of IgM memory B cells compared to responders and healthy controls.

The high degree of activation of B cells during HIV-1 infection may alter their response to activation signals The establishment of proper interaction at the site of immunological synapse between the T-B cells is pivotal for B cell activation and Ab production. This implies migration of Ag-stimulated B and T cells to secondary lymphoid organs [67] where differentiation of memory B cells and plasma cells occurs [68]. In the case of B cells responding to T cell independent Ags (Ags), these cells need to reach the marginal zone in the spleen and other secondary lymphoid organs. Thus the expression and function of chemokines and their receptors is pivotal for homing of B cells to the relevant compartments.

A. Cagigi et al. Little is known on chemokine receptor expression on B cells during HIV-1 infection. During physiological conditions, mature na¨ıve B cells (IgDbright /IgMdull ) are able to recognize the Ag and to organize germinal centre (GC) reactions thus structuring secondary follicles. Ag-primed B cells alter the chemokine receptor expression since BCR triggering leads to a decrease in CXCR5 expression followed by up-regulation of CCR7 [69], thus leading to re-localization of Ag-primed B cells to the border between T and B zones and further proliferation in the GC dark zone. Non-proliferating cells, including memory B cells, accumulate at the opposite pole of the GC known as the light zone [70]. Non-Ag-primed na¨ıve B cells are pushed aside and form the mantle zone. The inner part of the mantle zone, called lymphocytic corona, contains a T cell-zone; the outer part, visible mainly in the spleen, is called marginal zone (MZ) [70]. Ag-primed B cells up-regulate CD40, the costimulatory molecules CD80 and CD86 and present the Ag through MHC class II complexes to T cells of the same specificity in the T cell-zone which respond with the secretion of CD40L, IL-4 and IL-10 [70]. Following T cell activation, B cells up-regulate the expression of the enzyme activation-induced cytidine deaminase (AID) that triggers class switch recombination (CSR) and somatic hypermutations (SHM) on the immunoglobulin germline transcripts [71]. Post GC B cells will thereafter mature into memory B cells or plasma cells where CXCR4 expression is increased on long-term plasma cell precursors leaving the GC for the BM [72] but not mucosal sites [73]. On the other hand, memory B cells retain the high expression of CXCR5, after exit from the lymphoid tissue [70,72,74]. MZ B cells proliferate in loco and differentiate into shortlived, IgM or IgG producing plasma cells that mediate a first line defense against Ags [75]. Lymphocytic corona and MZ B cells, during the early phases of infection and before specific Abs are produced, secrete natural Abs, mostly IgM, which respond to Ags with low affinity and thereafter become IgM memory B cells [76—78]. Response against T-independent Ags also takes place in the MZ and may lead to short-lived, IgG producing plasma cells as well [70]. We have investigated the expression of chemokine receptors that are important for B cell migration within lymphoid tissue (CXCR5 and CCR7) and B cell homing to bone marrow (CXCR4) (Mowafi F, Cagigi A, et al., unpublished results). In HIV-1-infected patients the expression of CXCR5 on na¨ıve and memory B cells is decreased compared to healthy controls independently of CD4+ T cell counts, and this may interfere with the formation of GCs in lymphoid tissue [76]. Interestingly, HIV-1-infected patients have a significantly higher proportion of early plasma cells (CD19dim /CD38high ) in peripheral blood than controls (Fig. 2). In addition, these early plasma cells express less CXCR4 on the cell surface and it is possible that this anomalous pattern of chemokine receptors may lead to altered homing of plasma cells. It remains to be studied in relevant animal models of HIV-1 infection whether changes in the expression of chemokine receptors alter the homing of newly formed plasma cells to the bone marrow thus contributing to poor Ab production. It is interesting that the expression of chemokine receptors is so dramatically altered on B cells during HIV-1 infection. The molecular mechanism leading to this dysregulation should be identified since this could be a potential therapeutical target to improve Abs production during HIV-1

B cell dysfunctions and HIV-1 vaccine design

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Figure 2 Expression of chemokine receptors on blood plasma cells. After Ficoll-separation, PBMC were washed in PBS, before incubation with the appropriate Abs at RT for 20 min. 1 × 106 PBMC were used for each staining. The following Abs were used; CD19Cy-chrome, CD38-FITC and CXCR4-, CXCR5-, CXCR3- and CCR7-PE. After two additional washes, cells were fixed in PFA 1.5%/PBS before acqusition on a FACSscan (Becton Dickinson). Data was analysed using the CellQuest programme. In the upper panel, the proportion of early plasma cells CD19dim /CD38high in patients and controls is displayed. In the middle and lower panels, the individual chemokine receptor expression on gated CD19dim /CD38high is shown.

infection. However, it is also puzzling that the expression of so many other molecules like CD5, CD10, CD21, CD27, CD70, CD95, CD95L and LAIR-1 is dysregulated on B cells during HIV-1 infection; these molecule are markers of abnormally represented and functionally disturbed B cell subpopulations often increased in severely immunocompromised patients [7—9,80—82]. Such alterations are detected on both na¨ıve and memory cells from HIV-1-infected patients and may have detrimental effects on both the ability of B cells to initiate response to new Ags (including HIV-1 and vaccination Ags) and to maintain the protective Ab levels to previously met Ags. In a study previously conducted by us, we found that CD27-CD19+ B cells isolated from HIV-1-infected subjects produce IgG [8] and it has been described in many different context that PBMCs obtained from non-HAART-treated, HIV-1-infected subjects when cultured produce a large amount of immunoglobulins in vitro. These phenomena deserve further studies since they are probably mediated by unspecific activation stimuli leading to production of low

affinity, polyspecific Abs [19] and lack of B cell responses to viral and bacterial Ags. Activation through TLRs, a family of trans-membrane receptors that through recognition of microbial components trigger a cascade of intracellular signaling events leading to immune responses to microbes, is the focus of many current investigations. The work of several investigators have already shown that the TLR-9 receptor, which is the receptor for unmetilated CpG motives present on bacterial DNA [83], is highly expressed on normal and malignant B cells [84—85]. Increase in the cytoplasmic expression of TLR-9 on resting B lymphocytes correlated with an increased capacity of these cells to respond to CpG stimulation by proliferation [84]. CpG activation of memory B cells through TLR-9 leads to cellular proliferation and immunoglobulin production, a mechanism that has been suggested to play an important role for maintenance of serological memory in the absence of Ag exposure in man [16]. A recently published paper has shown that in addition to memory B cells, CpG stimulation through the TLR-9 induced also na¨ıve B cells to

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A. Cagigi et al. proliferate [85—86], a process which occurs in absence of exogenous cytokines and independently of the presence in culture of plasmacytoid dendritic cells. Controversial results have however been published on the possibility of activating human CD27-na¨ıve B cells [16,87] and it remains unsettled if these different results are due to different technical approaches in the preparation of pure na¨ıve B cells. It is unknown to which extent B cells express cytoplasmic TLR-9 during HIV-1 infection and if they can respond to CpG. We performed a pilot experiment and studied the production of IgG, IgA and IgM from B cells isolated from 5 HIV-1 chronically infected patients and 3 controls in response to CD40 and CpG activation (Fig. 3). Activation through CD40 lead to increased production of IgG, IgA and IgM in patients as compared to controls. Interestingly, the presence of CpG in culture stimulated a conspicuous production of IgM and IgA but not of IgG. These initial results on CpG mediated activation of B cells from HIV-1 infection suggest that CpG may play an important role if included in a local HIV-1 microbicide aimed at increasing local production of IgA and first line IgM Abs responses. Whether CpG could be used as an adjuvant for systemic vaccination of HIV-1-infected patients, remains to be investigated. The high level of activation detected on B cells from HIV-1-infected patients suggest however that intense stimulation by CpG may be detrimental in a situation whether these cells are already activated to produce a large amount of non-specific Abs.

Future prospectives

Figure 3 Production of immunoglobulins from B cell cultures upon different stimuli. Total IgM (A), IgA (B) and IgG (C) were measured in 9-day culture supernatants. Briefly, PBMCs [5 HIV-1-infected patients (median CD4 T cell count 470 cells/␮l; range 70—660; viral load <50 copies/ml in all patients), 3 controls] were cultured in 48 well-plates at a concentration of 1 × 106 cells/well with medium only or 1 ␮g/ml anti-CD40 mAb (Diaclone), 100 ng/ml IL-4 (ImmunoTools) and 50 ng/ml IL-10 (Peprotech) or 10 mg/ml CpG ODN 2006 (CpG B) (Microsynth). Thereafter, 96 well-plates (Corning Inc.) coated overnight at 4 ◦ C with 50 ␮l/well of goat anti-human IgM, or A or G (H+L) (Dako) diluted 1:1000 in Sodium Carbonate buffer 0.1 M were blocked using Ovalbumin for 2 h at RT. Triplicate samples of culture supernatants diluted in PBS-Tween were added to the plates and incubated overnight. Plates were then washed four times with PBS-Tween followed by the addition of HRPconjugated rabbit anti-human IgM, A or IgG (Dako) incubated for 1 h at room temperature. After a final wash, the plates were incubated with HRP substrate buffer until absorbances were read at 450 nm with an automated spectrophotometer (Labsystems Multiscan). Empty bars show results obtained from cell cultures established from the blood of HIV-1 patients while grey bars represent controls. Fold increase (patients versus controls) is indicated above the bars.

During HIV-1 infection several factors may have a role in promoting B cell dysregulation characterized by loss of memory B cells and impaired Ab production. Surprisingly, it has been described that different HIV-1 protein components may have opposite effect on events of CSR [88—89] and further studies are needed to unravel the cumulative effect of HIV-1 proteins on CSR in vivo. The concentration of IL-7 is increased in the serum of infected patients [90] and this protein has been previously shown to affect CXCR4 expression and HIV-1 replication. Recent studies conducted by us indicated that the expression of chemokine receptors is profoundly altered during HIV-1 infection and it was previously published that high levels of CXCL13 can be found in the serum of HIV-1-infected patients [91]. The correlation between the increase of serum IL-7 levels and the large number of immature/transitional B cells in the blood of HIV-1-infected patients is intriguing [92] and it remains to be verified whether IL-7 also alters the homing and migration of B cells to site relevant for B cell maturation and Ag presentation by affecting the expression of chemokine receptors. A picture is emerging where an altered expression of chemokine receptors on mature na¨ıve B cells might interfere with a correct migration and subsequent GC formation within the secondary follicles during HIV-1 infection (Mowafi F, Cagigi A, et al., unpublished results). These dysfunction in expression of chemokine receptors might have a role in the establishment of hypergammaglobulinemia seen in patients [8] impairing the ability of B cells to produce class switched specific Abs. This pathogenic scenario deserves attention as one possible target for novel therapies aimed at correcting and improv-

B cell dysfunctions and HIV-1 vaccine design ing B cell responses during HIV-1 infection. In addition if viral components play a role in dysregulated expression of chemokine receptors during HIV-1 infection, these HIV-1 proteins should be part of an HIV-1 vaccine formulation to ensure that the encounter with the virus following vaccination may not lead to B cell damage and loss of protective neutralizing Abs.

Acknowledgments The work of the investigators is supported through grants received from the Swedish MRC, the Swedish International Development Agency (SIDA-SAREC), the Swedish Children Cancer Foundation and the European Union (Europrise Network of Excellence). Financial support was also provided through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and the Karolinska Institute. AC is supported by a PhD grant from the Regione Autonoma della Sardegna.

References [1] Baba TW, Liska V, Hofmann-Lehmann R, Vlasak J, Xu W, Ayehunie S, et al. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat Med 2000;6(2):200—6. [2] Trkola A, Kuster H, Rusert P, Joos B, Fischer M, Leemann C, et al. Delay of HIV-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies. Nat Med 2005;11(6):615—22. [3] Hessell AJ, Hangartner L, Hunter M, Havenith CE, Beurskens FJ, Bakker JM, et al. Fc receptor but not complement binding is important in antibody protection against HIV. Nature 2007;449(7158):101—4. [4] Zolla-Pazner S. Improving on nature: focusing the immune response on the V3 loop. Hum Antibodies 2005;14(3/4):69— 72. [5] Dhillon AK, Donners H, Pantophlet R, Johnson WE, Decker JM, Shaw GM, et al. Dissecting the neutralizing antibody specificities of broadly neutralizing sera from human immunodeficiency virus type 1-infected donors. J Virol 2007;81(12):6548—62. [6] Zhou T, Xu L, Dey B, Hessell AJ, Van Ryk D, Xiang SH, et al. Structural definition of a conserved neutralization epitope on HIV-1 gp120. Nature 2007;445(7129):732—7. [7] De Milito A, Morch C, Sonnerborg A, Chiodi F. Loss of memory (CD27) B lymphocytes in HIV-1 infection. AIDS (London, England) 2001;15(8):957—64. [8] De Milito A, Nilsson A, Titanji K, Thorstensson R, Reizenstein E, Narita M, et al. Mechanisms of hypergammaglobulinemia and impaired antigen-specific humoral immunity in HIV-1 infection. Blood 2004;103(6):2180—6. [9] Moir S, Malaspina A, Pickeral OK, Donoghue ET, Vasquez J, Miller NJ, et al. Decreased survival of B cells of HIV-viremic patients mediated by altered expression of receptors of the TNF superfamily. J Exp Med 2004;200(5):587—99. [10] Terpstra FG, Al BJ, Roos MT, De Wolf F, Goudsmit J, Schellekens PT, et al. Longitudinal study of leukocyte functions in homosexual men seroconverted for HIV: rapid and persistent loss of B cell function after HIV infection. Eur J Immunol 1989;19(4):667—73. [11] Miedema F, Petit AJ, Terpstra FG, Schattenkerk JK, de Wolf F, Al BJ, et al. Immunological abnormalities in human immunodeficiency virus (HIV)-infected asymptomatic homosexual men. HIV affects the immune system before CD4+ T helper cell depletion occurs. J Clin Invest 1988;82(6):1908—14.

3023 [12] Titanji K, Chiodi F, Bellocco R, Schepis D, Osorio L, Tassandin C, et al. Primary HIV-1 infection sets the stage for important B lymphocyte dysfunctions. AIDS (London, England) 2005;19(17):1947—55. [13] Ochsenbein AF, Pinschewer DD, Sierro S, Horvath E, Hengartner H, Zinkernagel RM. Protective long-term antibody memory by antigen-driven and T help-dependent differentiation of longlived memory B cells to short-lived plasma cells independent of secondary lymphoid organs. In: Proceedings of the National Academy of Sciences of the United States of America, vol. 97(November (24)). 2000. p. 13263—8. [14] Slifka MK, Antia R, Whitmire JK, Ahmed R. Humoral immunity due to long-lived plasma cells. Immunity 1998;8(3):363—72. [15] Hammarlund E, Lewis MW, Hansen SG, Strelow LI, Nelson JA, Sexton GJ, et al. Duration of antiviral immunity after smallpox vaccination. Nat Med 2003;9(9):1131—7. [16] Bernasconi NL, Traggiai E, Lanzavecchia A. Maintenance of serological memory by polyclonal activation of human memory B cells. Science (New York) 2002;298(5601):2199—202. [17] Traggiai E, Puzone R, Lanzavecchia A. Antigen dependent and independent mechanisms that sustain serum antibody levels. Vaccine 2003;21(Suppl. 2):S35—7. [18] Nagase H, Agematsu K, Kitano K, Takamoto M, Okubo Y, Komiyama A, et al. Mechanism of hypergammaglobulinemia by HIV infection: circulating memory B-cell reduction with plasmacytosis. Clin Immunol (Orlando, FL) 2001;100(2): 250—9. [19] Titanji K, De Milito A, Cagigi A, Thorstensson R, Grutzmeier S, Atlas A, et al. Loss of memory B cells impairs maintenance of long-term serologic memory during HIV-1 infection. Blood 2006;108(5):1580—7. [20] D’Orsogna LJ, Krueger RG, McKinnon EJ, French MA. Circulating memory B-cell subpopulations are affected differently by HIV infection and antiretroviral therapy. AIDS (London, England) 2007;21(13):1747—52. [21] Hart M, Steel A, Clark SA, Moyle G, Nelson M, Henderson DC, et al. Loss of discrete memory B cell subsets is associated with impaired immunization responses in HIV-1 infection and may be a risk factor for invasive pneumococcal disease. J Immunol 2007;178(12):8212—20. [22] Kroon FP, van Dissel JT, Ravensbergen E, Nibbering PH, van Furth R. Antibodies against pneumococcal polysaccharides after vaccination in HIV-infected individuals: 5-year follow-up of antibody concentrations. Vaccine 1999;18(5/6):524—30. [23] Kroon FP, van Dissel JT, Ravensbergen E, Nibbering PH, van Furth R. Impaired antibody response after immunization of HIV-infected individuals with the polysaccharide vaccine against Salmonella typhi (Typhim-Vi). Vaccine 1999;17(23/24): 2941—5. [24] Laursen AL, Andersen PL. Low levels of IgG antibodies against Pneumocystis carinii among HIV-infected patients. Scand J Infect Dis 1998;30(5):495—9. [25] Pitzurra L, Perito S, Baldelli F, Bistoni F, Vecchiarelli A. Humoral response against Cryptococcus neoformans mannoprotein antigens in HIV-infected patients. Clin Exp Immunol 2003;133(1):91—6. [26] Bekker V, Westerlaken GH, Scherpbier H, Alders S, Zaaijer H, van Baarle D, et al. Varicella vaccination in HIV-1-infected children after immune reconstitution. AIDS (London, England) 2006;20(18):2321—9. [27] Janoff EN, O’Brien J, Thompson P, Ehret J, Meiklejohn G, Duvall G, et al. Streptococcus pneumoniae colonization, bacteremia, and immune response among persons with human immunodeficiency virus infection. J Infect Dis 1993;167(1):49—56. [28] French N, Moore M, Haikala R, Kayhty H, Gilks CF. A case-control study to investigate serological correlates of clinical failure of 23-valent pneumococcal polysaccharide vaccine in HIV-1infected Ugandan adults. J Infect Dis 2004;190(4):707—12.

3024 [29] Malaspina A, Moir S, Orsega SM, Vasquez J, Miller NJ, Donoghue ET, et al. Compromised B cell responses to influenza vaccination in HIV-infected individuals. J Infect Dis 2005;191(9):1442—50. [30] de Moraes-Pinto MI, Farhat CK, Carbonare SB, Curti SP, Otsubo ME, Lazarotti DS, et al. Maternally acquired immunity in newborns from women infected by the human immunodeficiency virus. Acta Paediatr 1993;82(12):1034—8. [31] Embree JE, Datta P, Stackiw W, Sekla L, Braddick M, Kreiss JK, et al. Increased risk of early measles in infants of human immunodeficiency virus type 1-seropositive mothers. J Infect Dis 1992;165(2):262—7. [32] Moss WJ, Monze M, Ryon JJ, Quinn TC, Griffin DE, Cutts F. Prospective study of measles in hospitalized, human immunodeficiency virus (HIV)-infected and HIV-un-infected children in Zambia. Clin Infect Dis 2002;35(2):189—96. [33] Scott S, Cumberland P, Shulman CE, Cousens S, Cohen BJ, Brown DW, et al. Neonatal measles immunity in rural Kenya: the influence of HIV and placental malaria infections on placental transfer of antibodies and levels of antibody in maternal and cord serum samples. J Infect Dis 2005;191(11):1854—60. [34] Helfand RF, Moss WJ, Harpaz R, Scott S, Cutts F. Evaluating the impact of the HIV pandemic on measles control and elimination. Bull World Health Organ 2005;83(5):329—37. [35] Chong Y, Ikematsu H, Yamamoto M, Murata M, Yamaji K, Nishimura M, et al. Increased frequency of CD27- (naive) B cells and their phenotypic alteration in HIV type 1-infected patients. AIDS Res Hum Retroviruses 2004;20(6):621—9. [36] Zhang ZQ, Casimiro DR, Schleif WA, Chen M, Citron M, Davies ME, et al. Early depletion of proliferating B cells of germinal center in rapidly progressive simian immunodeficiency virus infection. Virology 2007;361(2):455—64. [37] Zinkernagel RM. On immunological memory. Philos Trans Royal Soc London 2000;355(1395):369—71. [38] Gaines H, von Sydow M, Sonnerborg A, Albert J, Czajkowski J, Pehrson PO, et al. Antibody response in primary human immunodeficiency virus infection. Lancet 1987;1(8544):1249—53. [39] Burton DR. Antibodies, viruses and vaccines. Nat Rev 2002;2(9):706—13. [40] Fenyo EM, Putkonen P. Broad cross-neutralizing activity in serum is associated with slow progression and low risk of transmission in primate lentivirus infections. Immunol Lett 1996;51(1/2):95—9. [41] Letvin NL, Barouch DH, Montefiori DC. Prospects for vaccine protection against HIV-1 infection and AIDS. Annu Rev Immunol 2002;20:73—99. [42] Mascola JR. Defining the protective antibody response for HIV1. Curr Mol Med 2003;3(3):209—16. [43] Cecilia D, Kleeberger C, Munoz A, Giorgi JV, Zolla-Pazner S. A longitudinal study of neutralizing antibodies and disease progression in HIV-1-infected subjects. J Infect Dis 1999;179(6):1365—74. [44] Morris L, Binley JM, Clas BA, Bonhoeffer S, Astill TP, Kost R, et al. HIV-1 antigen-specific and -nonspecific B cell responses are sensitive to combination antiretroviral therapy. J Exp Med 1998;188(2):233—45. [45] Notermans DW, de Jong JJ, Goudsmit J, Bakker M, Roos MT, Nijholt L, et al. Potent antiretroviral therapy initiates normalization of hypergammaglobulinemia and a decline in HIV type 1-specific antibody responses. AIDS Res Hum Retroviruses 2001;17(11):1003—8. [46] Fondere JM, Huguet MF, Yssel H, Baillat V, Reynes J, van de Perre P, et al. Detection of peripheral HIV-1-specific memory B cells in patients untreated or receiving highly active antiretroviral therapy. AIDS (London, England) 2003;17(16): 2323—30. [47] Kroon FP, Rimmelzwaan GF, Roos MT, Osterhaus AD, Hamann D, Miedema F, et al. Restored humoral immune response

A. Cagigi et al.

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61] [62]

[63]

to influenza vaccination in HIV-infected adults treated with highly active antiretroviral therapy. AIDS (London, England) 1998;12(17):F217—23. Deayton JR, Sabin CA, Britt WB, Jones IM, Wilson P, Johnson MA, et al. Rapid reconstitution of humoral immunity against cytomegalovirus but not HIV following highly active antiretroviral therapy. AIDS (London, England) 2002;16(16):2129—35. Melvin AJ, Mohan KM. Response to immunization with measles, tetanus, and Haemophilus influenzae type b vaccines in children who have human immunodeficiency virus type 1 infection and are treated with highly active antiretroviral therapy. Pediatrics 2003;111(6 Pt 1):e641—4. Jordano Q, Falco V, Almirante B, Planes AM, del Valle O, Ribera E, et al. Invasive pneumococcal disease in patients infected with HIV: still a threat in the era of highly active antiretroviral therapy. Clin Infect Dis 2004;38(11):1623—8. French M, Keane N, McKinnon E, Phung S, Price P. Susceptibility to opportunistic infections in HIV-infected patients with increased CD4 T cell counts on antiretroviral therapy may be predicted by markers of dysfunctional effector memory CD4 T cells and B cells. HIV Med 2007;8(3):148—55. Janoff EN, Hardy WD, Smith PD, Wahl SM. Humoral recall responses in HIV infection. Levels, specificity, and affinity of antigen-specific IgG. J Immunol 1991;147(7):2130—5. Kroon FP, van Dissel JT, de Jong JC, van Furth R. Antibody response to influenza, tetanus and pneumococcal vaccines in HIV-seropositive individuals in relation to the number of CD4+ lymphocytes. AIDS (London, England) 1994;8(4):469—76. Arpadi SM, Markowitz LE, Baughman AL, Shah K, Adam H, Wiznia A, et al. Measles antibody in vaccinated human immunodeficiency virus type 1-infected children. Pediatrics 1996;97(5):653—7. Janoff EN, Douglas Jr JM, Gabriel M, Blaser MJ, Davidson AJ, Cohn DL, et al. Class-specific antibody response to pneumococcal capsular polysaccharides in men infected with human immunodeficiency virus type 1. J Infect Dis 1988;158(5):983—90. King Jr JC. Community respiratory viruses in individuals with human immunodeficiency virus infection. Am J Med 1997;102(3A):19—24 [discussion 5—6]. Fuller JD, Craven DE, Steger KA, Cox N, Heeren TC, Chernoff D. Influenza vaccination of human immunodeficiency virus (HIV)-infected adults: impact on plasma levels of HIV type 1 RNA and determinants of antibody response. Clin Infect Dis 1999;28(3):541—7. Kroon FP, van Dissel JT, de Jong JC, Zwinderman K, van Furth R. Antibody response after influenza vaccination in HIV-infected individuals: a consecutive 3-year study. Vaccine 2000;18(26):3040—9. Moss WJ, Scott S, Mugala N, Ndhlovu Z, Beeler JA, Audet SA, et al. Immunogenicity of standard-titer measles vaccine in HIV-1infected and un-infected Zambian children: an observational study. J Infect Dis 2007;196(3):347—55. Lange CG, Lederman MM, Medvik K, Asaad R, Wild M, Kalayjian R, et al. Nadir CD4+ T cell count and numbers of CD28+ CD4+ T cells predict functional responses to immunizations in chronic HIV-1 infection. AIDS (London, England) 2003;17(14):2015—23. Lesinski GB, Westerink MA. Vaccines against polysaccharide antigens. Curr Drug Targets 2001;1(3):325—34. Rodriguez-Barradas MC, Musher DM, Lahart C, Lacke C, Groover J, Watson D, et al. Antibody to capsular polysaccharides of Streptococcus pneumoniae after vaccination of human immunodeficiency virus-infected subjects with 23-valent pneumococcal vaccine. J Infect Dis 1992;165(3):553—6. Rodriguez-Barradas MC, Groover JE, Lacke CE, Gump DW, Lahart CJ, Pandey JP, et al. IgG antibody to pneumococcal capsular polysaccharide in human immunodeficiency virus-infected subjects: persistence of antibody in

B cell dysfunctions and HIV-1 vaccine design

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74] [75]

[76]

[77] [78]

responders, revaccination in nonresponders, and relationship of immunoglobulin allotype to response. J Infect Dis 1996;173(6):1347—53. Ahmed F, Steinhoff MC, Rodriguez-Barradas MC, Hamilton RG, Musher DM, Nelson KE. Effect of human immunodeficiency virus type 1 infection on the antibody response to a glycoprotein conjugate pneumococcal vaccine: results from a randomized trial. J Infect Dis 1996;173(1):83—90. Kroon FP, van Dissel JT, Ravensbergen E, Nibbering PH, van Furth R. Enhanced antibody response to pneumococcal polysaccharide vaccine after prior immunization with conjugate pneumococcal vaccine in HIV-infected adults. Vaccine 2000;19(7/8):886—94. Subramaniam KS, Segal R, Lyles RH, Rodriguez-Barradas MC, Pirofski LA. Qualitative change in antibody responses of human immunodeficiency virus-infected individuals to pneumococcal capsular polysaccharide vaccination associated with highly active antiretroviral therapy. J Infect Dis 2003;187(5):758—68. Cyster JG. Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs. Annu Rev Immunol 2005;23:127—59. McHeyzer-Williams LJ, McHeyzer-Williams MG. Antigenspecific memory B cell development. Annu Rev Immunol 2005; 23:487—513. Casamayor-Pallej` a M, Mondi` ere P, Verschelde C, Bella C, Defrance T. BCR ligation reprograms B cells for migration to the T zone and B-cell follicle sequentially. Blood 2002; 99(6):1913—21. Sagaert X, Sprangers B, De Wolf-Peeters C. The dynamics of the B follicle: understanding the normal counterpart of B-cellderived malignancies. Leukemia 2007;21(7):1378—86. Pan-Hammarstrom Q, Zhao Y, Hammarstrom L. Class switch recombination: a comparison between mouse and human. Advances Immunol 2007;93:1—61. Avery DT, Ellyard JI, Mackay F, Corcoran LM, Hodgkin PD, Tangye SG. Increased expression of CD27 on activated human memory B cells correlates with their commitment to the plasma cell lineage. J Immunol 2005;174(7):4034—42 [erratum in: J Immunol 2005;174((May 1)9):5885]. Kunkel EJ, Kim CH, Lazarus NH, Vierra MA, Soler D, Bowman EP, et al. CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells. J Clin Invest 2003;111(7):1001—10. Tarlinton D. B-cell memory: are subsets necessary? Nat Rev 2006;6(10):785—90. Sagaert X, De Wolf-Peeters C. Classification of B cells according to their differentiation status, their micro-anatomical localisation and their developmental lineage. Immunol Lett 2003;90(2/3):179—86. Carsetti R, Rosado MM, Donnanno S, Guazzi V, Soresina A, Meini A, et al. The loss of IgM memory B cells correlates with clinical disease in common variable immunodeficiency. J Allergy Clin Immunol 2005;115(2):412—7. Carsetti R, Rosado MM, Wardmann H. Peripheral development of B cells in mouse and man. Immunol Rev 2004;197:179—91. Alugupalli KR, Leong JM, Woodland RT, Muramatsu M, Honjo T, Gerstein RM. B1b lymphocytes confer T cell-independent longlasting immunity. Immunity 2004;21(3):379—90.

3025 [80] Wolthers KC, Otto SA, Lens SM, Van Lier RA, Miedema F, Meyaard L. Functional B cell abnormalities in HIV type 1 infection: role of CD40L and CD70. AIDS Res Hum Retroviruses 1997;13(12):1023—9. [81] Moir S, Malaspina A, Ogwaro KM, Donoghue ET, Hallahan CW, Ehler LA, et al. HIV-1 induces phenotypic and functional perturbations of B cells in chronically infected individuals. In: Proceedings of the National Academy of Sciences of the United States of America, vol. 98(August (18)). 2001. p. 10362—7. [82] Titanji K, Nilsson A, Morch C, Samuelsson A, Sonnerborg A, Grutzmeier S, et al. Low frequency of plasma nerve-growth factor detection is associated with death of memory B lymphocytes in HIV-1 infection. Clin Exp Immunol 2003;132(2):297—303. [83] Bauer S, Kirschning CJ, Hacker H, Redecke V, Hausmann S, Akira S, et al. Human TLR-9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. In: Proceedings of the National Academy of Sciences of the United States of America, vol. 98(July (16)). 2001. p. 9237—42. [84] Bourke E, Bosisio D, Golay J, Polentarutti N, Mantovani A. The toll-like receptor repertoire of human B lymphocytes: inducible and selective expression of TLR-9 and TLR-10 in normal and transformed cells. Blood 2003;102(3):956—63. [85] Jiang W, Lederman MM, Harding CV, Rodriguez B, Mohner RJ, Sieg SF. TLR-9 stimulation drives naive B cells to proliferate and to attain enhanced antigen presenting function. Eur J Immunol 2007;37(8):2205—13. [86] Huggins J, Pellegrin T, Felgar RE, Wei C, Brown M, Zheng B, et al. CpG DNA activation and plasma-cell differentiation of CD27-naive human B cells. Blood 2007;109(4):1611—9. [87] Bernasconi NL, Onai N, Lanzavecchia A. A role for Toll-like receptors in acquired immunity: up-regulation of TLR-9 by BCR triggering in naive B cells and constitutive expression in memory B cells. Blood 2003;101(11):4500—4. [88] Qiao X, He B, Chiu A, Knowles DM, Chadburn A, Cerutti A. Human immunodeficiency virus 1 Nef suppresses CD40dependent immunoglobulin class switching in bystander B cells. Nat Immunol 2006;7(3):302—10. [89] He B, Qiao X, Klasse PJ, Chiu A, Chadburn A, Knowles DM, et al. HIV-1 envelope triggers polyclonal Ig class switch recombination through a CD40-independent mechanism involving BAFF and C-type lectin receptors. J Immunol 2006;176(7): 3931—41. [90] Rethi B, Fluur C, Atlas A, Krzyzowska M, Mowafi F, Grutzmeier S, et al. Loss of IL-7Ralpha is associated with CD4 T cell depletion, high interleukin-7 levels and CD28 downregulation in HIV infected patients. AIDS (London, England) 2005;19(18):2077—86. [91] Widney DP, Breen EC, Boscardin WJ, Kitchen SG, Alcantar JM, Smith JB, et al. Serum levels of the homeostatic B cell chemokine, CXCL13, are elevated during HIV infection. J Interferon Cytokine Res 2005;25(11):702—6. [92] Malaspina A, Moir S, Ho J, Wang W, Howell ML, O’Shea MA, et al. Appearance of immature/transitional B cells in HIV-infected individuals with advanced disease: correlation with increased IL-7. In: Proceedings of the National Academy of Sciences of the United States of America, vol. 103(February (7)). 2006. p. 2262—7.