Low-affinity immunoglobulin (Ig)G with potential autoreactivity to lymphocytes and hypergammaglobulinaemia have been described previously in HIV-1-infected patients. Whether such antibodies increase after challenging the immune system, for example with an immunization, is not known. In the present study, the modulation of antibodies with low affinity and potential autoreactivity was evaluated after 2012–13 seasonal flu vaccination with a simple empirical laboratory test measuring the titres of anti-lymphocyte antibodies (ALA) in two different models of secondary immunodeficiency: HIV-1 vertically infected patients (HIV) and patients treated with immunosuppressive therapies after kidney transplantation (KT) compared to healthy individuals (HC). In parallel, the activation status of B cells and their degree of immune senescence was evaluated by measuring the B cell interleukin (IL)-21R expression/plasma IL-21 levels and the frequencies of mature-activated (MA) and double-negative (DN) B cells. A significant increase of ALA titres was observed after vaccination in HIV and KT but not in HC, and this correlated directly with the frequencies of both MA and DN and inversely with the B cell IL-21R expression. This suggests that the quality of an immune response triggered by flu vaccination in HIV and KT may depend upon the activation status of B cells and on their degree of immune senescence. Further investigations are needed to verify whether high frequencies of MA and DN may also relate to increase autoimmunity after immunization in high-risk populations.
The ability of B cells to differentiate into antibody-secreting cells that produce high-affinity antibodies is the key for a successful immune response upon vaccination . Terminal differentiation of B cells and hypergammaglobulinaemia are hallmarks of B cell hyperactivity in human immune deficiency virus (HIV)-1 disease [2, 3]. In addition, the presence of an altered subpopulation of CD27– B cells expressing switched immunoglobulins (Ig) was reported in HIV-1-infected individuals . Phenotypically, this B cell subpopulation resembles the double-negative (CD27–IgD–) (DN) B cells found at high frequencies in the blood of healthy elderly individuals . Another subpopulation of B cells phenotypically similar to the ones described above is the mature-activated (CD10–CD21–) (MA), which has been related to the degree of chronic immune-activation in viraemic HIV-1-infected patients . Furthermore, it has been shown previously, as in conditions of chronic pathological immune stimulation, that B cells produced IgG, known as anti-lymphocyte antibodies (ALA) or polyspecific self-reactive antibodies (PSA), which retain low-affinity characteristics with a spectrum of antigens, including self-antigens [7-9]. These conditions have been reported in cases of long-term systemic exposure to a self-antigen, for example in systemic lupus erythematosus (SLE)  or long-term exposure to infectious agents, such as during HIV-1 infection [11, 12]. Whether ALA can also be detected in patients with solid organ transplantation has never been investigated. As during HIV-1 infection, patients with solid organ transplantation may in fact also experience chronic immune activation due to the persistence of a foreign antigen (in this case the transplanted organ) and are immune-deficient because of the immunosuppressive therapies administered after transplantation. Non-specifically activated B cells should not be capable of increasing antibody affinity to a given antigen through immunization. However, it is likely that high levels of ALA can be produced upon vaccination in those patients with chronic immune activation. We tested this hypothesis in the present study. The modulation of antibodies with low affinity and potential autoreactivity was evaluated after immunization with a simple empirical laboratory test measuring the titres of ALA [11, 13] in two different models of secondary immunodeficiency: HIV-1 vertically infected patients and kidney-transplanted patients under treatment with immunosuppressive therapies. In parallel, the levels of ALA were analysed in relation to signs of premature ageing of the B cell compartment, such as DN and MA B cell subpopulations.
Materials and methods
A total of 65 anti-retroviral therapy (ART)-treated HIV-1 vertically infected patients (abbreviated as HIV), 81 patients undergoing immunosuppressive therapy following kidney transplantation (abbreviated as KT) and 23 healthy controls of similar age (abbreviated as HC) were enrolled between September 2012 and November 2012 at the Bambino Gesù Children's Hospital, Rome, Italy. KT are usually treated by means of a triple immunosuppressive schedule: a calcineurine inhibitor, cyclosporin or tacrolimus (usually cyclosporin as a first line and tacrolimus following rejection), mycophenolate mofetil (600 mg/m2 twice a day (b.i.d.) in cyclosporin-treated patients or 300 mg/m2 b.i.d. in association with tacrolimus) and steroids (low-dose prednisone every second day, 0·1–0·2 mg/kg/every other day). Written informed consent was obtained from all subjects or parents/legal guardians before enrolment and the ethical committees of the Bambino Gesù Children's Hospital approved the study. Characteristics of all subjects are summarized in Table 1.
Table 1. Characteristics of the subjects.
KT: kidney transplantations; HC: healthy controls; s.d.: standard deviation.
No. of subjects
65 (27 males, 38 females)
81 (49 males, 32 females)
23 (13 males, 10 females)
Age mean (± s.d.)
14·3 (± 6·5)
17·2 (± 6·0)
13·3 (± 9·6)
HIV-1 viral load median (range)
50 (<50–21 700) Copies/ml
CD4+ T cell counts median (range)
Vaccination and sample preparation
All subjects received one dose of the inactivated influenza vaccine trivalent types A and B (Split Virion) VAXIGRIP® (Sanofi Pasteur, Maidenhead, UK) during October and November 2012. Blood was collected prior to vaccination and after 21 days from vaccination. Peripheral blood mononuclear cells (PBMCs) and plasma were purified from Ficoll (LiStarFish, Milan, Italy) ethylenediamine tetraacetic acid (EDTA)-treated blood and temporarily frozen until further analyses.
Detection of ALA
Detection of ALA was performed as described previously . Briefly, PBMC from a healthy donor were purified from a buffy-coat and washed three times with fresh phosphate-buffered saline (PBS) for 10 min (at 180 g, 146 g and 115 g) in order to minimize the amount of thrombocytes, and subsequently incubated in complete RPMI for 30 min at 37°C in order to remove the fraction of monocytes adhering to the flask bottom. Cells were subsequently seeded in a 96-well plate at a concentration of 0·5 × 106 cells/well, washed with 1% bovine serum albumin (BSA)–PBS and incubated with 100 μl plasma samples for 1 h on ice. After another wash step with 1% BSA–PBS, PBMC were incubated with a F(ab′)2 goat polyclonal fluorescein isothiocyanate (FITC)-conjugated anti-human IgG-F(ab′)2 cross-adsorbed antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA) for 30 min on ice and finally washed with 1% BSA–PBS. Multi-colour flow cytometry was performed on a fluorescence activated cell sorter (FACS)Canto, interfaced to a FacsDiva software (BD Biosciences, San Jose, CA, USA) and analysed through Flow-Jo software version 8·8·3 (Three Star Inc., Ashland, OR, USA). The binding of the antibody to the cells incubated with the different plasma samples was measured and the percentage of binding-inhibition calculated according to the background staining (cells incubated without plasma). A cartoon showing the principles of the assay is presented in Fig. 1.
Purified PBMCs were thawed and stained with the following conjugated monoclonal antibodies: CD19-Alexa 488, interleukin (IL)-21R-phycoerythrin (PE), CD27-peridinin chlorophyll-cyanin 5·5 (PerCP-Cy5·5), CD21-allophycocyanin (APC), IgD-H7 (all from BD Biosciences) and the CD10-PE-Cy7 (Biolegend, San Diego, CA, USA). The frequencies of MA (defined as CD10–CD21–) and DN (defined as CD27–IgD–) B cell subpopulations were calculated from total CD19+ B cells. Multi-colour flow cytometry was performed on a FACSCanto, interfaced to a FacsDiva software (BD Biosciences) and analysed through Flow-Jo software version 8·8·3 (Three Star Inc.).
Enzyme-linked immunosorbent assay (ELISA)
Plasma IL-21 titres were measured using the human IL-21 platinum ELISA kit (eBioscience, San Diego, CA, USA), following the manufacturer's instructions.
The Mann–Whitney U-test and Spearman's correlation were used for all analyses. A P-value <0·05 was considered statistically significant. GraphPad Prism software for Windows was used to perform the analyses.
ALA modulation after flu immunization in the different patient groups
The ALA titres before and after flu vaccination were quantitated as described in the Materials and methods and in Fig. 1. Before vaccination, significantly lower ALA titres were found in the HIV group compared to KT and HC (P < 0·0001) (Fig. 2a), while no significant difference was found between the KT and the HC groups (P > 0·05) (Fig. 2a). Interestingly, after vaccination individuals in both the HIV and KT groups increased ALA titres substantially compared to HC (P = 0·0001 and P = 0·0002, respectively) (Fig. 2b). Between HIV and KT, the biggest increase was recorded in the HIV group (P = 0·0008) (Fig. 2c). HC increased ALA titres only slightly compared to HIV and KT (P = 0·0001 and P = 0·0003, respectively (Fig. 2c). Fifteen per cent of the HIV-1-infected individuals (10 of 65) were having a viraemic blip at the time of vaccination (Table 1). However, this did not relate to any of the parameters analysed as confirmed by Spearman's correlation (P > 0·05). Moreover, the CD4+ T cell counts were similar in the viramic and aviraemic patients (P > 0·05).
B cell immune activation and senescence in the different patient groups
It has been reported recently that the up-regulation of IL-21 receptor (R) on B cells and of plasma IL-21 levels could distinguish among A(H1N1)pdm09 vaccine responders and non-responders . Thus, in order to assess whether the ALA increase observed in the HIV and KT groups after flu immunization, related to a different activation status of B cells or to a different degree of immune senescence in these groups, the B cell IL-21R expression and the frequencies of mature-activated (CD10–CD21–) (MA) and double-negative (CD27–IgD–) (DN) B cells were measured in parallel to plasma IL-21 levels. The levels of IL-21R expression on B cells was significantly higher in the HC group compared to HIV and KT (P < 0·0001), with the lowest level observed in the HIV group compared to KT (P = 0·02) (Fig. 3a). A similar scenario was observed for the plasma IL-21 levels, where the HC presented with higher levels compared to HIV and KT (P < 0·0001 and P = 0·008, respectively) (Fig. 3b). Interestingly, the lowest levels of plasma IL-21 were recorded in the KT group (P = 0·01 in comparison with HIV) (Fig. 3b). Conversely, the frequencies of both MA and DN were significantly higher in both the HIV and KT groups compared to HC (P < 0·0001 for both HIV and KT versus HC for MA and P = 0·0005 and P = 0·002, respectively, for DN) (Fig. 3c,d). The gating strategy for the identification of MA and DN is shown in Fig. 4.
B cell immune activation and senescence in relation to ALA titres before and after flu immunization
While dividing the patients between individuals who did not increase (Delta−) and increased (Delta+) the ALA titres after flu immunization, it appears clear that higher B cell IL-21R expression was present prior to vaccination in those individuals belonging to the Delta– group (P = 0·004) (Fig. 5a). The plasma IL-21 levels were not significantly higher in the Delta– group compared to the Delta+ (P = 0·08) (Fig. 5b). An opposite scenario was observed for the frequencies of both MA and DN that were significantly higher before vaccination in the Delta+ group (P = 0·0009 and P = 0·001, respectively) (Fig. 5c,d). In line with the data shown in Fig. 5, while a significant direct correlation was observed between the ALA titres and the B cell IL-21R expression before vaccination (r = 0·2/P = 0·004), this reversed after vaccination (r = −0·2/P = 0·002) (Fig. 6a,b). The plasma IL-21 levels correlated with the ALA titres both prior to and after vaccination (r = 0·2/P = 0·001 and r = 0·2/P = 0·03) (Fig. 6a,b). Moreover, the frequencies of both MA and DN correlated directly with the ALA titres after vaccination (r = 0·2/P = 0·007 and r = 0·2/P = 0·001, respectively) (Fig. 6c). Finally, while the frequencies of MA correlated directly with the B cell IL-21R expression (r = 0·2/P = 0·002) this was not the case for the frequencies of DN, where a strong inverse correlation was observed (r = −0·5/P < 0·0001) (Fig. 6d).
ALA have been detected previously during HIV-1 infection and been shown to bind lymphocytes mediating T cell death . Accordingly, it was described later that HIV-1 infection is often characterized by increased levels of total IgG (hypergammaglobulinaemia) of low affinity and potential autoreactivity . The modulation of such antibodies after challenging the immune system with vaccination has never been investigated.
While the need and effectiveness of flu vaccination in HC is still debated , seasonal flu vaccination is recommended for HIV-1-infected individuals  and for patients with other immune disorders featuring loss of protective immunity, such as patients undergoing immunosuppressive therapy following solid organ transplantation [18-20].
In the present work, the modulation of ALA after 2012–13 seasonal flu vaccination was evaluated in two different cohorts of patients with acquired immunodeficiency due to HIV-1 infection (HIV) or due to immunosuppressive therapy following kidney transplantation (KT) compared to healthy individuals (HC). Before vaccination, no significant differences in ALA titres were found between the KT and the HC groups. However, after vaccination individuals in both the HIV and KT groups increased ALA titres significantly compared to HC, who had only a slight increase.
Chronic immune-activation during HIV-1 infection has been shown to lead to B cell exhaustion and death in parallel to increased frequencies of MA [6, 21]. Moreover, a B cell subpopulation similar to the DN found in elderly individuals  has been reported in HIV-1-infected patients . Therefore, the frequencies of MA and DN in parallel to the B cell IL-21R expression and plasma IL-21 levels were investigated in relation to the ALA modulation after vaccination in all individuals. Both the HIV and the KT groups presented lower levels of B cell IL-21R expression and plasma IL-21 with higher levels of MA and DN compared to HC. This suggests that individuals included in the HIV and in the KT groups may have a similar status of B cell activation and, despite being mainly adolescents, a similar degree of immune senescence, possibly accounting for the premature ageing of their immune system. In support of this, it has been reported recently that even lymphocytes from children infected with HIV-1 have short telomeres . Similarly, lower teleromase activity has been detected in lymphocytes from long-term survivors of kidney transplantation . This provides further evidence that immune senescence may occur in these populations. No such data are available for patients under immunosuppressive therapy. Interestingly, among all individuals who did not increase (Delta−) the ALA titres after vaccination, higher levels of B cell IL-21R expression and plasma IL-21 with lower levels of MA and DN were observed compared to individuals who had an increased (Delta+). Moreover, while a direct correlation was found between B cell IL-21R expression and ALA titres before vaccination, this reversed after vaccination, thus reinforcing the positive role indicated previously for the B cell IL-21R during vaccination . The frequencies of both MA and DN correlated directly with the ALA titres after vaccination and inversely to B cell IL-21R expression.
Together, the results of the present study suggest that the quality of a humoral immune response triggered by vaccination in HIV and KT may depend upon the activation status of B cells and on their degree of immune senescence. Increased MA and DN may account for the abnormal increase of ALA titres observed after immunization in these populations. Further investigations are needed to confirm this hypothesis and to investigate further the role of such antibodies, and whether high frequencies of MA and DN may also relate to increase autoimmunity after immunization in high-risk populations.
We wish to thank all the personnel at the Bambino Gesù Children's Hospital who helped in coordinating vaccination. We wish to thank Miss Jennifer Faudella for her administrative work.