Antiretroviral therapy (ART) has dramatically reduced the morbidity and mortality of HIV-infected patients, whereas HIV-specific immune responses remain unchanged, even after years of successful viral control. Indeed, HIV-1 replication rapidly rebounds after ART withdrawal to levels similar to that observed before ART initiation.1 Therapeutic immunization strategies have been proposed to enhance immune-specific responses that could allow ART discontinuation over time.1 Depletion of CD4 T-cell counts and dysfunction of CD4 helper and CD8 cytotoxic T cells result in an ineffective HIV-specific response.2,3 Cytotoxic CD8 T cells affect viral control by directly killing infected cells and enhancing global immune response by cytokine secretion. Indeed, the HIV-specific effector CD8 T cells are more frequently detectable in a rare group of patients who can control HIV replication in the absence of treatment (HIV-1 elite controllers) than in regular HIV-1 progressors.4 Thus, therapeutic strategies that stimulate and enhance CD8 T-cell responses may have a potential role in the control of viral replication.
Dendritic cell (DC)–based therapeutic vaccination in simian immunodeficiency virus and HIV infections is one strategy that has shown encouraging results.1 At least 10 clinical trials have provided evidence that DC-based immunotherapy in infected individuals can elicit HIV-specific immunological responses. Some of these studies reported reductions in levels of viral replication when HIV-specific responses were elicited.1,5,6 Monocyte-derived DCs loaded with aldrithiol-2–inactivated autologous virus stimulated proliferative responses on autologous CD4 and CD8 T cells in HIV-infected patients. In addition, proviral DNA, viral loads, and HIV-1 RNA levels were significantly decreased in autologous T cells expanded with virus-pulsed DCs.7 Our group has shown a partial to complete restoration of HIV-specific proliferative immune responses in successfully ART-treated HIV-infected patients receiving autologous (personalized) DC-based vaccine (AGS-004). This vaccine was produced from autologous monocyte-derived DCs electroporated with RNA encoding CD40 ligand (CD40L) and patients' own HIV antigens collected before beginning ART.8 Here, we evaluated the impact of DC immunotherapy on additional immune parameters that include peripheral T- and B-cell memory subsets and levels of immune activation in patients receiving AGS-004 DC vaccine therapy.
MATERIALS AND METHODS
Ten ART-treated subjects with HIV plasma RNA below the level of detection (<50 copies/mL), median CD4 T-cell count of 440 cells per microliter (range: 316–1102), and with a CD4 T-cell nadir >200 cells per microliter were studied at the McGill University Health Centre, Montreal, Canada, as previously described.8,9 Ethical approval and written informed consent from all subjects were obtained before study initiation (Clinical Trial Registry number NCT00381212).
AGS-004 was manufactured at Argos Therapeutics Inc., Durham, NC according to current good manufacturing practice as previously described.8,9 Briefly, monocytes were isolated from the standard leukapheresis (COBE Spectra Apheresis System; CaridianBCT, Lakewood, CO) and cultured with granulocyte macrophage colony-stimulating factor and interleukin 4 for 5 days to generate DCs. These DCs were matured overnight by adding tumor necrosis factor α, interferon γ, and prostaglandin E2 to the cultures. The resulting mature DCs were electroporated on day 7 with autologous RNA of HIV antigens (Gag, Vpr, Rev, and Nef) amplified from HIV RNA in the subject's pre-ART plasma and poly-adenylated CD40L RNA. The final AGS-004 product was suspended in 10% dimethyl sulfoxide and 10% dextrose for injection in autologous plasma, quality control tested for sterility, and cryopreserved as individual doses of 1.2 × 107 DCs. Eligible subjects received intradermal injections of AGS-004 every 4 weeks for 4 treatments (weeks 0, 4, 8, and 12) to a single axillary lymph node area.8–10
B- and T-Cell Subset Assessment by Flow Cytometry
Changes in B- and T-cell subsets were assessed on the peripheral blood mononuclear cells obtained at week 0 and week 14 (2 weeks after AGS-004 immunotherapy). Anti-CD5-PerCP-Cy5.5, CD10-PE-Cy7, CD-19-APC-Cy7, CD27-Alexa700, CD27-PE, CD38-Alexa700, CD38-APC, immunoglobulin D (IgD)-FITC, CD138–APC, CD3-PacBleu, CD4-APC-Cy7, CD4-PerCP-CY5.5, CCR7-PE-Cy7, CD25-PE, CD127-PE-Cy7, PD-1-FITC, HLA-DR-PE, BCL2-FITC, and Ki67-PE were obtained from BD Biosciences (Mississauga, ON, Canada). Anti-CD45RA-ECD and CD23-ECD were obtained from Beckman Coulter (Mississauga, ON, Canada). Anti-CD8-APC-Eflour780 and FoxP3-Alexa488 were obtained from eBiosciences (San Diego, CA). Vivid was obtained from Invitrogen (Burlington, ON, Canada). Cells were analyzed on an LSR II (BD Immunocytometry Systems, San Jose, CA). After gating on single live cells (Vividlow cells), CD4+ and CD8+ subsets were characterized as naive (CD45RA+CCR7+CD27+), central memory (CD45RA−CCR7+CD27+), transitional memory (CD45RA−CCR7−CD27+), effector memory (CD45RA−CCR7−CD27−), and terminally differentiated (CD45RA+CCR7−CD27−). Regulatory T cells (Tregs) were characterized as CD3+CD4+CD25highFoxP3highCD127low. B-lymphocyte subsets were characterized as naive B cells Bm1 (CD19+CD38−IgD+CD27−CD10−CD23−CD5−CD138−), activated naive B cells Bm2 (CD19+CD38+IgD+CD27−CD10−CD23+CD5−CD138−), germinal central founder cells Bm2′(CD19+CD38+IgD+CD27−CD10+CD23−CD5−CD138−), centroblasts and centrocytes Bm3 and Bm4 (CD19+CD38+IgD−CD27−CD10+CD23−CD5−CD138−), early memory B cells eBm5 (CD19+CD38+IgD−CD27+CD10−CD23−CD5−CD138−), memory B cells Bm5 (CD19+CD38−IgD−CD27+ CD10−CD23−CD5−CD138−), IgD memory B cells (CD19+CD38−IgD+CD27+CD10−CD23−CD5−CD138−), plasma cells (CD19+CD38+IgD−CD27+CD10−CD23−CD5−CD138+), and B-1 cells (CD19+CD38−IgD−CD27−CD10−CD23−CD5+CD138−).
Statistical analyses were assessed using the paired nonparametric Wilcoxon signed rank test (GraphPad Prism 5.0 statistical software).
Autologous DC-Based Immunotherapy Induces Bm1 Naive B-Cell Proliferation
We first evaluated the impact of DC-based immunotherapy on phenotypic characteristics of B cells in patients continuing to receive ART after DC immunization. The changes were measured before (week 0) and 2 weeks after the 4 axillary injections of AGS-004 (week 14). The frequency of total, naive, and memory B-cell counts remained unchanged after DC-based immunotherapy [median (range) 3.37% (1.16–7.57) vs. 5.1% (2.24–7.78), P = 0.27, for total B cells, 61.2% (41.9–77.2) vs. 61.95% (39.9–86.4), P = 0.9, for CD19+CD27− naive B cells, and 33.1% (13.5–53.1) vs. 31.5% (10.1–56.3), P = 0.85, for CD19+CD27+ memory B cells; data not shown]. Among B-cell subsets (Fig. 1A), the percentages of Bm2′, Bm3, Bm4, eBm5, Bm, B1, IgD memory, and plasma cells were not changed after DC-based immunotherapy (Figs. 1B, C; P > 0.05 for all comparisons). The percentage of Bm2 cells was moderately decreased [median (range) 2.36% (0.6–7.55) vs. 2.27% (0.39–9.76), P = 0.048], whereas there was a 2-fold increase in Bm1 naive T cells [median (range) 3.85% (0.46–8.81) vs. 5.42% (0.82–20.7), P = 0.019] (Fig. 1B, 1C). To assess whether the Bm1 cell increase was linked to cell proliferation or to increased survival, we studied the expressions of the proliferation marker Ki67 and the anti-apoptotic marker BCL-2 on B cells. Our results showed an increase in the expression of Ki67 on B cells after DC-based immunotherapy [median (range) 21.7% (9.28–42.6) vs. 20.9% (16.2–49.5), P = 0.02] (Fig. 1D) in the absence of changes in BCL-2 expression [median (range) 13.7% (6.1–24.5) vs. 12.2% (6.75–34.5), P = 0.37] (Fig. 1E).
Autologous DC-Based Immunotherapy Does Not Affect Treg Expansion and General Immune Activation
The frequency of total CD4 and CD8 T cells did not change after DC-based immunotherapy [median (range) 39.7% (19.3–44.5) vs. 31.7% (21.3–52) and 27.5% (15.5–42.8) vs. 25% (12.3–43.4), respectively, P < 0.05; data not shown]. Percentages of total, naive, central memory, transitional memory, effector memory, terminally differentiated T-cell subsets and Tregs did not change after DC-based immunotherapy (Figs. 2A–D). Levels of CD38/HLA-DR/PD-1 expression on CD4 and CD8 T cells also remained unchanged, suggesting the absence of induction of immune activation at the time of the study measurements (Fig. 2E).
We have assessed the tolerance and B- and T-cell subsets in HIV-infected subjects receiving an autologous DC-based immunotherapy where HIV replication was controlled by ART. Ten subjects received immunotherapy at weekly intervals for 4 weeks with blood samples collected before immunotherapy and 2 weeks after the last administration of AGS-004 to assess memory B- and T-cell subset changes. AGS-004 is designed to present in vitro transcribed RNA encoding specific autologous HIV antigens (Gag, Vpr, Rev, and Nef), along with a synthetically derived RNA CD40L to achieve DC maturation.8,9 The use of autologous pre-ART plasma HIV as the source of the antigens addresses the problem of the extreme genetic diversity in HIV when using consensus or reference HIV protein sequences as immunogens.10 It has been previously shown that ex vivo cytokine-induced maturation of DCs followed by electroporation with RNA encoding CD40L proteins, along with the RNA antigen payload, can improve the immunopotency of the final DC product11 and may induce polyclonal immune responses.8,10,11 CD40 is expressed on a variety of cells, including monocytes, DCs, endothelial cells, and epithelial cells. Although its ligand is expressed mainly on activated CD4 helper T cells, it can also be found on other cells. CD40L plays a crucial role in priming other immune cells.12,13 Therefore, B cells and DCs may directly interact via cell contact–dependent mechanisms such as CD40–CD40L independent of CD4 T-cell help.13 On naive B cells, cross-linking of CD40 promotes B-cell survival, proliferation, and differentiation.12 Our results showed a significant increase of Bm1 naive cells after AGS-004 immunotherapy. To further evaluate the reason for this increase, we evaluated Ki67 and intracellular BCL-2 expressions14,15 as markers of proliferation and survival, respectively.16 There was a significant increase of Ki67 expression by B cells after AGS-004 immunotherapy in the absence of BCL-2 expression, indicating a DC-induced B-cell proliferation at the time of measurement. Binding of CD40 by CD40L helps to drive the resting B cell into the cell cycle, which is essential for B-cell responses.17 Indeed, CD40-stimulated B cells pulsed with antigens are effective Ig-secreting or antigen-presenting cells that can generate specific T cells.18,19 In addition, plasmacytoid DCs may induce polyclonal naive B-cell expansion and B-cell differentiation toward Ig-producing cells via type I interferon-enhanced TLR7 sensitivity of B cells.20 Further longitudinal studies are needed to assess the evolution/differentiation of newly generated naive B cells after AGS-004 DC-based immunotherapy.
We then evaluated DC immunotherapy on CD4 and CD8 T-cell subsets, immune activation, and Treg induction. The absolute CD4 and CD8 counts and the frequency of CD4 and CD8 memory subsets did not change after DC immunotherapy. It has been suggested that HIV-specific CD4 T-cell activation after HIV-1 recombinant canarypox (ALVAC-HIV) therapeutic immunization may be the reason for the vaccine failure and can render patients more susceptible to disease progression upon skewed induction of activated CD4 T-cell targets.21 Because AGS-004 is electroporated by encoding specific HIV antigens (Gag, Vpr, Rev, and Nef), induction of immune activation might be expected. We therefore evaluated the expression of activation markers such as the combination of HLA-DR, CD38, and PD-1 on CD4 and CD8 T cells after AGS-004 immunotherapy. Interestingly, our results showed no significant changes in the expression of these T-cell immune activation markers. The abrogation of CD40–CD40L signaling pathway impedes the homeostasis of thymic resident Tregs by decreasing interleukin 2 expression.22 Furthermore, monocyte-derived DCs from breast cancer tumors are biased to induce Treg responses that can impede DC-based therapeutic approaches for cancer patients.23 Tregs play an immunosuppressive role in HIV pathogenesis by inhibition of T-cell proliferation and anti–HIV-specific immune responses.24,25 Macatangay et al26 have reported a mild increase of Treg frequency associated with a lower Gag-specific polyfunctional CD8 T-cell response in ART-treated subjects who received a therapeutic vaccine consisting of autologous DCs loaded with HIV-1 peptides. Indeed, Tregs may suppress HIV-specific polyfunctional responses induced by DC-based vaccines.26 Of interest, our results showed that AGS-004 immunotherapy did not affect Treg frequency suggesting its immunogenicity.
Altogether, our findings showed that DC immunotherapy induced naive B-cell proliferation without accompanying T-cell changes, including subsets and Treg frequency, associated with the absence of any clinical adverse events or autoimmune antibody changes as we previously reported.8 Furthermore, no changes in plasma levels of total IgG, IgM, or IgA Igs were observed during the study (data not shown). A larger longitudinal trial would help to overcome some limitations of this study that include the following: (1) only a small number of HIV-infected subjects participated in this study, (2) the 18-week follow-up period may not be long enough to observe significant changes of B-cell subsets, and (3) possible confounding variables, such as HIV-specific antibody production and cytokine levels, were not evaluated.
This study indicates that AGS-004 did not induce changes in the proportion of CD4 and CD8 T-cell subsets, including Treg, and it did not induce immune activation. We observed a 2-fold increase in the frequency of naive B cells (Bm1) in the absence of increased memory long-lived B cells or plasma cells. The study findings pave the way to conduct larger studies using AGS-004 as a specific inducer of HIV CD8 T-cell responses8 in the absence of changes in T-cell immune activation markers.
The authors are grateful to Dr Mohamed-Rachid Boulassel for his helpful participation in the study design and his technical assistance and advice. The authors are thankful to Jacquie Sas and Jim Pankovich from Canadian Institutes of Health Research Canadian HIV Trials Network for study implementation and coordination and to Angie Massicotte and Kishanda Vyboh for technical assistance.
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