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ISB 1442 is a fully human bispecific antibody (BsAb) using BEAT (Bispecific Engagement by Antibodies based on the T-cell receptor) platform to target CD38 and CD47 as treatment for CD38+ malignancies, including multiple myeloma (MM). ISB 1442 is designed with a bi-paratopic anti-CD38 arm that strongly binds to CD38+ tumor cells and an anti-CD47 arm made of a single Fab designed to block interaction between CD47 and the signal-regulatory protein alpha (SIRPα) with low affinity. This approach is expected to induce minimal unintended effects on red blood cells (RBC) compared to anti-CD47 monoclonal antibody (mAb) magrolimab as it enables the CD47 binding only upon avidity induced CD38 crosslinking. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). In vitro, ISB 1442 exhibited higher killing potency compared to daratumumab across a range of CD38-expressing tumor cells. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, acting mostly through ADCP. To assess the complex mechanisms of action of ISB 1442 in a single system, a multiple mode of action of killing (MMoAK) assay was established to allow for simultaneous killing by natural killer cells, autologous macrophages, as well as complement from human serum. In the MMoAK assay, ISB 1442 exhibited tumor cell killing that was twice as high as daratumumab. In vivo, in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice, ISB 1442 induced higher tumor growth inhibition than daratumumab and comparable tumor regression to magrolimab. On-target specificity was evaluated in vitro in human and monkey whole blood assays. ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation and showed markedly lower RBC hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile in humans. On the contrary, due to the higher expression of CD38 in monkey RBC compared to human, ISB 1442 showed more pronounced binding on RBC than magrolimab, suggesting that monkey is a more sensitive species than human for toxicological evaluation of CD38-targeted therapies. Finally, by integrating in vitro pharmacology data along with available clinical information on benchmark antibodies, a quantitative systems pharmacology model was developed to simulate potential efficacious dose range in MM. In summary, we report a novel approach for the treatment for CD38+ cancers by co-targeting CD38 and CD47. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity by overcoming known primary and acquired tumor escape mechanisms of resistance relative to daratumumab. Citation Format: Stefano Sammicheli, Camille Grandclement, Elie Dheilly, Maria Panagopoulou, Evangelia Martini, Perrine Suere, Blandine Pouleau, Carole Estoppey, Julia Frei, Jeremy Loyau, Thierry Monney, Adam Drake, Alain Rubod, Marie Agnes Doucey, Vinu Menon, Venkatesha Udupa, Sunitha GN, Daniel Rasmussen, Jeppe Koch Olsen, Roberto Gionannini, Girish Gudi, Ankita Srivastava, Cyril Konto, Mario Perro. ISB 1442, a first-in-class CD38 and CD47 bispecific antibody innate cell modulator for the treatment of CD38+ malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2903.

Diffuse large B cell lymphomas (DLBCL) are a highly heterogeneous subtype of Non Hodgkin Lymphoma (NHL), accounting for about 25% of NHL [1]. Despite an increased progression-free survival upon therapy, 40-50% of patients develop relapse/refractory disease, therefore there remains an important medical need [2]. T cell recruiting therapies, such as the CD20xCD3 T cell bi-specific antibody CD20-TCB (RG6026 or glofitamab), represent a novel approach to target all stages of DLBCL, especially those that fail to respond to multiple lines of treatment [3, 4]. We aimed for a better understanding of the molecular features related to the mode of action (MoA) of CD20-TCB in inducing Target/T cell synapse formation and human T cell recruitment to the tumor. To directly evaluate the correlation between synapse, cytokine production and anti-tumor efficacy using CD20-TCB, we developed an innovative preclinical human DLBCL in vivo model that allowed tracking in vivo human T cell dynamics by multiphoton intravital microscopy (MP-IVM). By ex vivo and in vivo approaches, we revealed that CD20-TCB is inducing strong and stable synapses between human T cell and tumor cells, which are dependent on the dose of CD20-TCB and on LFA-1 activity but not on FAS-L. Moreover, despite CD20-TCB being a large molecule (194.342 kDa), we observed that intra-tumor CD20-TCB-mediated human T cell-tumor cell synapses occur within 1 hour upon CD20-TCB administration. These tight interactions, observed for at least 72 hours post TCB administration, result in tumor cell cytotoxicity, resident T cell proliferation and peripheral blood T cell recruitment into tumor. By blocking the IFNγ-CXCL10 axis, the recruitment of peripheral T cells was abrogated, partially affecting the efficacy of CD20-TCB treatment which rely only on resident T cell proliferation. Altogether these data reveal that CD20-TCB’s anti-tumor activity relies on a triple effect: i) fast formation of stable T cell-tumor cell synapses which induce tumor cytotoxicity and cytokine production, ii) resident T cell proliferation and iii) recruitment of fresh peripheral T cells to the tumor core to allow a positive enhancement of the anti-tumor effect.

Introduction Cancer Immune Therapies have shown unprecedented results in improving tumor control.1–3 However, many patients are still refractory to treatment. A deeper understanding of the mode of action of the different CITs sub-classes may help improving therapeutic approaches to reach better anti-tumor response. For this reason, we developed a multi-photon intra-vital microscopy (MP-IVM) approach to study in vivo, at single cell level, the tumor microenvironment upon treatment with CD20-targeting T-cell bispecific antibodies (TCB) [4] in a preclinical model of diffuse large B cell lymphomas (DLBCL). Methods To selectively monitor clinical lead molecules in the context of human T cell responses, we developed a skinfold chamber model [5] in last generation humanized mice [6] that allows visualization, by MP-IVM, of labelled human T cells co-injected intra-dermally with WSU-DLCL2, a human DLBCL. We have used this model to investigate T cells recruitment to tumors upon CD20-TCB therapy: by intra-venously injecting labeled T cells in mice treated with selected blocking antibodies, we were able to identify dedicated pathways induced by CD20-TCB and regulating T cell influx into the tumor bed. Furthermore, we developed a user-independent quantification platform to assess changes in the dynamics of T cell motility and time of interaction with tumor cells. Results/Discussion We have developed an experimental preclinical model that aims to reduce xenoreaction (human T cell reaction against mouse tissue) by utilizing T cells derived from humanized mice, educated within murine thymus. We demonstrate that such model is optimal to quantify human T cell dynamics in vivo. We show that CD20-TCB localizes in the tumor and acts on tumor-resident T cell motility within 1 hour post i.v. injection (defined as functional PK), causing a sharp reduction in their speed (from 4 to 2 µm/min) and an increase in tumor/T cell interaction time; those changes last up to 72h post-treatment. In addition, we prove how the initial tumor/T cell interaction mediated by CD20-TCB lead to peripheral T cells recruitment into the tumor. This mechanism is dependent on the presence of tumor-resident T cells and on IFNg-CXCL10 pathway. Inhibiting any of these two parameters resulted in reduced T cells infiltration from the periphery and reduced anti-tumor efficacy. Conclusion We developed a reliable imaging and analysis pipeline to investigate in vivo T cell dynamics and recruitment and applied it to the study of CD20-TCB treatment of DLBCL model. Our approach has shed new lights into the MoA of this new class of immune-therapeutics, demonstrating that the IFNγ-CXCL10 pathway is involved in T cell recruitment upon CD20-TCB treatment. Disclosure Information F. Cremasco: A. Employment (full or part-time); Significant; Roche Glycart AG. E. Menietti: A. Employment (full or part-time); Significant; Roche Glycart AG. D. Speziale: A. Employment (full or part-time); Significant; Roche Glycart AG. J. Sam: A. Employment (full or part-time); Significant; Roche Glycart AG. S. Sammicheli: A. Employment (full or part-time); Significant; Roche Glycart AG. S. Chen: A. Employment (full or part-time); Significant; Roche Glycart AG. M. Richard: A. Employment (full or part-time); Significant; Roche Glycart AG. C. Klein: A. Employment (full or part-time); Significant; Roche Glycart AG. P. Umana: A. Employment (full or part-time); Significant; Roche Glycart AG. M. Bacac: A. Employment (full or part-time); Significant; Roche Glycart AG. S. Colombetti: A. Employment (full or part-time); Significant; Roche Glycart AG. M. Perro: A. Employment (full or part-time); Significant; Roche Glycart AG.

PD-L1/PD-1 blocking antibodies have demonstrated therapeutic efficacy across a range of human cancers. Extending this benefit to a greater number of patients, however, will require a better understanding of how these therapies instigate anticancer immunity. Although the PD-L1/PD-1 axis is typically associated with T cell function, we demonstrate here that dendritic cells (DCs) are an important target of PD-L1 blocking antibody. PD-L1 binds two receptors, PD-1 and B7.1 (CD80). PD-L1 is expressed much more abundantly than B7.1 on peripheral and tumor-associated DCs in patients with cancer. Blocking PD-L1 on DCs relieves B7.1 sequestration in cis by PD-L1, which allows the B7.1/CD28 interaction to enhance T cell priming. In line with this, in patients with renal cell carcinoma or non-small cell lung cancer treated with atezolizumab (PD-L1 blockade), a DC gene signature is strongly associated with improved overall survival. These data suggest that PD-L1 blockade reinvigorates DC function to generate potent anticancer T cell immunity.

Summary Cross-presentation of antigens from dead tumor cells by type 1 conventional dendritic cells (cDC1s) is thought to underlie priming of anti-cancer CD8+ T cells. cDC1 express high levels of DNGR-1 (a.k.a. CLEC9A), a receptor that binds to F-actin exposed by dead cell debris and promotes cross-presentation of associated antigens. Here, we show that secreted gelsolin (sGSN), an extracellular protein, decreases DNGR-1 binding to F-actin and cross-presentation of dead cell-associated antigens by cDC1s. Mice deficient in sGsn display increased DNGR-1-dependent resistance to transplantable tumors, especially ones expressing neoantigens associated with the actin cytoskeleton, and exhibit greater responsiveness to cancer immunotherapy. In human cancers, lower levels of intratumoral sGSN transcripts, as well as presence of mutations in proteins associated with the actin cytoskeleton, are associated with signatures of anti-cancer immunity and increased patient survival. Our results reveal a natural barrier to cross-presentation of cancer antigens that dampens anti-tumor CD8+ T cell responses., Graphical abstract, Highlights • Secreted gelsolin (sGSN) inhibits DNGR-1 binding to F-actin • sGSN dampens DNGR-1-dependent cross-presentation of dead cell-associated antigens • sGSN impairs DNGR-1-dependent cDC1-mediated anti-tumor immunity • Low sGSN expression and mutations in FABPs correlate with cancer patient survival, The secreted gelsolin component of the plasma actin-scavenging system impairs the ability of the receptor DNGR-1 to recognize dead cells and selectively dampens cross-presentation of tumor antigens by type 1 dendritic cells, acting as a barrier to anti-tumor immunity.

Humoral immune responses depend on B cells encountering antigen (Ag) in lymph nodes (LNs) draining infection sites, getting activated, interacting with different cells, proliferating and differentiating into antibody (Ab)-secreting cells. Each of these events occurs in distinct LN sub-compartments, requiring the migration of B cells from niche to niche in a fast and tightly coordinated fashion. While some of the rules that characterize B cell behavior in secondary lymphoid organs have been elucidated at the population level, we have only limited knowledge of the precise dynamics of B cell interactions with different kinds of LN cells at the single-cell level. Here, we describe in detail an intravital microscopy technique that allows the analysis of B cell dynamic behavior in the popliteal lymph node of anesthetized mice at high spatial and temporal resolution. A detailed understanding of the spatiotemporal dynamics of B cells within secondary lymphoid organs may lead to novel, rational vaccine strategies aimed at inducing rapid and long-lived humoral immune responses.

Vaccination is one of the most cost-effective health interventions and, with the exception of water sanitization, no other action has had such a major effect in mortality reduction. Combined with other approaches, such as clean water, better hygiene, and health education, vaccination contributed to prevent millions of cases of deaths among children under 5 years of age. New or improved vaccines are needed to fight some vaccine-preventable diseases that are still a threat for the public health globally, as reported also in the Global Vaccine Action Plan (GVAP) endorsed by the World Health Assembly in 2012. Adjuvants are substances that enhance the effectiveness of vaccination, but despite their critical role for the development of novel vaccines, very few of them are approved for use in humans. Aluminum hydroxide (Alum) is the most common adjuvant used in vaccines administered in millions of doses around the world to prevent several dangerous diseases. The development of an improved version of Alum can help to design and produce new or better vaccines. Alum/toll-like receptor (TLR)7 is a novel Alum-based adjuvant, currently in phase I clinical development, formed by the attachment of a benzonaphthyridine compound, TLR7 agonist, to Alum. In preclinical studies, Alum/TLR7 showed a superior adjuvant capacity, compared to Alum, in several disease models, such as meningococcal meningitis, anthrax, staphylococcus infections. None of these studies reported the effect of Alum/TLR7 on the generation of the B cell memory compartment, despite this is a critical aspect to achieve a better immunization. In this study, we show, for the first time, that, compared to Alum, Alum/TLR7 enhances the expansion of the memory B cell compartment within the draining lymph node (LN) as result of intranodal sustained proliferation of antigen-engaged B cells and/or accumulation of memory B cells. In addition, we observed that Alum/TLR7 induces a recruitment of naïve antigen-specific B cells within the draining LN that may help to sustain the germinal center reaction. Our data further support Alum/TLR7 as a new promising adjuvant, which might contribute to meet the expectations of the GVAP for 2020 and beyond.

Conventional type 1 dendritic cells (cDC1) are critical for antitumor immunity, and their abundance within tumors is associated with immune-mediated rejection and the success of immunotherapy. Here, we show that cDC1 accumulation in mouse tumors often depends on natural killer (NK) cells that produce the cDC1 chemoattractants CCL5 and XCL1. Similarly, in human cancers, intratumoral CCL5, XCL1, and XCL2 transcripts closely correlate with gene signatures of both NK cells and cDC1 and are associated with increased overall patient survival. Notably, tumor production of prostaglandin E2 (PGE2) leads to evasion of the NK cell-cDC1 axis in part by impairing NK cell viability and chemokine production, as well as by causing downregulation of chemokine receptor expression in cDC1. Our findings reveal a cellular and molecular checkpoint for intratumoral cDC1 recruitment that is targeted by tumor-derived PGE2 for immune evasion and that could be exploited for cancer therapy.

Humoral immune responses depend on B cells encountering antigen (Ag) in lymph nodes (LNs) draining infection sites, getting activated, interacting with different cells, proliferating and differentiating into antibody (Ab)-secreting cells. Each of these events occurs in distinct LN sub-compartments, requiring the migration of B cells from niche to niche in a fast and tightly coordinated fashion. While some of the rules that characterize B cell behavior in secondary lymphoid organs have been elucidated at the population level, we have only limited knowledge of the precise dynamics of B cell interactions with different kinds of LN cells at the single-cell level. Here, we describe in detail an intravital microscopy technique that allows the analysis of B cell dynamic behavior in the popliteal lymph node of anesthetized mice at high spatial and temporal resolution. A detailed understanding of the spatiotemporal dynamics of B cells within secondary lymphoid organs may lead to novel, rational vaccine strategies aimed at inducing rapid and long-lived humoral immune responses.

Bisphosphonates are a class of drugs that are widely used to inhibit loss of bone mass in patients. We show here that the administration of clinically relevant doses of bisphosphonates in mice increases antibody responses to live and inactive viruses, proteins, haptens and existing commercial vaccine formulations. Bisphosphonates exert this adjuvant-like activity in the absence of CD4+ and γδ T cells, neutrophils or dendritic cells and their effect does not rely on local macrophage depletion nor does it depend upon Toll-like receptor signaling or the inflammasome. Rather, bisphosphonates target directly B cells and enhance B cell expansion and antibody production upon antigen encounter. These data establish bisphosphonates as a novel class of adjuvants that boost humoral immune responses.

Antibodies are critical for protection against viral infections. However, several viruses, such as lymphocytic choriomeningitis virus (LCMV), avoid the induction of early protective antibody responses by poorly understood mechanisms. We analyzed the spatiotemporal dynamics of B cell activation to show that, upon subcutaneous infection, LCMV-specific B cells readily relocate to the interfollicular and T cell areas of draining lymph nodes, where they extensively interact with CD11b+Ly6Chi inflammatory monocytes. These myeloid cells were recruited to lymph nodes draining LCMV infection sites in a type I interferon– and CCR2-dependent fashion, and they suppressed antiviral B cell responses by virtue of their ability to produce nitric oxide. Depletion of inflammatory monocytes, inhibition of their lymph node recruitment, or impairment of their nitric oxide–producing ability enhanced LCMV-specific B cell survival and led to robust neutralizing antibody production. Our results identify inflammatory monocytes as critical gatekeepers that restrain antiviral B cell responses and suggest that certain viruses take advantage of these cells to prolong their persistence within the host.

Ruffin N, Lantto R, Pensieroso S, Sammicheli S, Hejdeman B, Rethi B, Chiodi F (Karolinska Institutet, Stockholm; and South Hospital, Stockholm, Sweden). Immune activation and increased IL-21R expression are associated with the loss of memory B cells during HIV-1 infection. J Intern Med 2012; 272: 492–503. Objectives. Microbial translocation and chronic immune activation were previously shown to be associated with impairment of T cell functions and disease progression during infection with human immunodeficiency virus type-1 (HIV-1); however, their impact on B cell function and number remains unknown. By measuring markers of immune activation and molecules involved in apoptosis regulation, we have evaluated the association between microbial translocation and loss of memory B cells in HIV-1-infected patients. Methods. Markers of activation [the interleukin-21 receptor (IL-21R) and CD38] and apoptosis (Bim, Bcl-2 and annexin V) were measured in B cell subpopulations by multicolour flow cytometry. Levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS), measures of microbial translocation, were determined in plasma. Purified B cells were also exposed in vitro to Toll-like receptor (TLR) ligands. Results. IL-21R expression was higher in cells from HIV-1-infected patients, compared with control subjects, with the highest levels in nontreated patients. An inverse correlation was observed between IL-21R expression and percentages of circulating resting memory (RM) B cells. IL-21R-positive memory B cells were also more susceptible to spontaneous apoptosis and displayed lower levels of Bcl-2. It is interesting that the levels of sCD14, which are increased during HIV-1 infection, were correlated with decreased percentages of RM B cells and high IL-21R expression. In the plasma of HIV-1-infected individuals, a correlation was found between sCD14 and LPS levels. TLR activation of B cells in vitro resulted in IL-21R up-regulation. Conclusions. Microbial translocation and the associated immune activation during HIV-1 infection may lead to high expression levels of the IL-21R activation marker in RM B cells, a feature associated with increased apoptosis and a reduced number of these cells in the circulation.

Interleukin-7 (IL-7) promotes the maintenance and activation of peripheral T cells, whereas it does not act directly on mature B cells due to the lack of IL-7Rα expression on these. We report here that, in spite of the insensitivity of B cells to IL-7, high concentration of IL-7 can lead to increased B cell survival and antibody production in the presence of T cells, without the use of any further B cell stimulatory signal. IL-7 promoted B cell activation through inducing CD70 expression on resting T cells, particularly on CD4+ memory cells. The interaction of CD70 molecules with the B cell costimulatory receptor CD27 led to B cell proliferation, the accumulation of CD38 + CD20- plasmablasts and antibody production. In addition, IL-7 treatment induced BAFF secretion from resting peripheral T cells thereby promoting B cell survival. IL-7 levels can increase in lymphopenic conditions, in autoimmune diseases or in patients receiving T cell regenerative IL-7 therapy. Based on our findings high IL-7 levels can lead to increased B cell activation by inducing the B cell regulatory proteins CD70 and BAFF in resting T cells. Such activity might be beneficial in short term immune-stimulatory IL-7 therapies; permanently increased IL-7 levels, on the other hand, can contribute to impaired B cell tolerance.

Background. CD28 - T lymphocytes progressively increase during aging, autoimmunity, and HIV-1 infection. Expansion of these cells stands in contrast with their senescent phenotype described by several studies. Understanding the functional properties and phenotype of CD28 - T cell during HIV-1 infection is important, because this subset incorporates T cells specific for HIV-1 and other chronic pathogens.Methods. Blood samples were obtained from 23 healthy and 43 HIV-1–infected individuals: 26 receiving antiretroviral therapy and 17 naive to treatment. The phenotype of CD28 - and CD28 + T cells was determined by flow cytometry. T cells were activated through T-cell receptor before apoptosis and proliferation measurements. Interleukin (IL)–2, tumor-necrosis factor, interferon-γ, and perforin production were analyzed using enzyme-linked immunosorbent assay.Results. CD28 - T cells from patients receiving antiretroviral therapy exhibited a low sensitivity to apoptosis and enhanced proliferation after TCR stimulation, compared with T cells of uninfected individuals. On the contrary, CD28 - T cells from viremic patients showed a decreased Bcl-2 expression, a high sensitivity to apoptosis, and poor proliferative ability, compared with treated patients and control subjects. T cells from untreated patients produced less IL-2, possibly underlying their decreased proliferative abilities.Conclusions. The level of HIV-1 replication and associated immunoactivation represent a critical factor in regulating survival and activation of CD28 - T cells.

CD70 molecules expressed by activated T cells provide potent B cell stimulatory signals. We hypothesized that an altered CD70 expression might contribute to B cell abnormalities during HIV-1 infection.CD70 expression and the functional and migratory properties of the CD4CD70 T lymphocytes were analyzed in HIV-1-infected patients and in humanized mice. Correlations were tested between CD70 expression and features of B-cell activation, apoptosis sensitivity and functional exhaustion.CD4CD70 T cells were analyzed in cohorts of CD4 T-cell lymphopenic, viremic or nonlymphopenic, nonviremic HIV-1-infected patients and in noninfected individuals. CD70 upregulation was also followed in HIV-1-infected humanized mice. CD38, CD95, LAIR1 and PD-1 expressions were monitored on B-cell subpopulations, Ki67 was assessed to estimate B-cell proliferation and antibody levels were measured in plasma.Blood CD4CD70 T-cell frequencies increased in response to CD4 T-cell depletion or high viremia levels as a possible consequence of increased activation and proliferation in this subset. CD4CD70 T cells produced T-helper 1-type cytokines and expressed chemokine receptors mobilizing toward sites of inflammation but not to lymphoid follicles. High CD70 expression was observed in HIV-1-infected humanized mice at extrafollicular sites (peritoneum, bone-marrow). CD4CD70 T-cell frequencies correlated with the expression of the activation marker CD38 and the death receptor CD95 on various memory B-cell subsets, with B-cell proliferation and with plasma IgG levels.CD4CD70 T cells may contribute to B cell hyperactivation and accelerated memory B-cell turnover during HIV-1 infection.

SummaryEffector CD8+ T cells (CD8 TE) play a key role during hepatotropic viral infections. Here, we used advanced imaging in mouse models of hepatitis B virus (HBV) pathogenesis to understand the mechanisms whereby these cells home to the liver, recognize antigens, and deploy effector functions. We show that circulating CD8 TE arrest within liver sinusoids by docking onto platelets previously adhered to sinusoidal hyaluronan via CD44. After the initial arrest, CD8 TE actively crawl along liver sinusoids and probe sub-sinusoidal hepatocytes for the presence of antigens by extending cytoplasmic protrusions through endothelial fenestrae. Hepatocellular antigen recognition triggers effector functions in a diapedesis-independent manner and is inhibited by the processes of sinusoidal defenestration and capillarization that characterize liver fibrosis. These findings reveal the dynamic behavior whereby CD8 TE control hepatotropic pathogens and suggest how liver fibrosis might reduce CD8 TE immune surveillance toward infected or transformed hepatocytes.

The relevance of CD4+T-cells, viral load and age in the immunological response to influenza infection and vaccination in HIV-1 infected individuals has previously been pointed out. Our study aimed at assessing, in the setting of 2009 A(H1N1)pdm09 influenza vaccination, whether quantification of activation-induced deaminase (AID) expression in blood B-cells may provide additional indications for predicting antibody response to vaccination in HIV-1 infected patients with similar CD4+T-cell counts and age. Forty-seven healthy controls, 37 ART-treated and 17 treatment-naïve HIV-1 infected patients were enrolled in the study. Blood was collected prior to A(H1N1)pdm09 vaccination and at 1, 3 and 6 months after vaccination. Antibody titers to A(H1N1)pdm09 vaccine were measured by hemagglutination inhibition (HI) assay while the mRNA expression levels of AID were measured by quantitative real time PCR. Upon B-cell activation in vitro, AID increase correlated to antibody response to the A(H1N1)pdm09 vaccine at 1 month after vaccination in all individuals. In addition, the maximum expression levels of AID were significantly higher in those individuals who still carried protective levels of A(H1N1)pdm09 antibodies after 6 months from vaccination. No correlation was found between CD4+T-cell counts or age at vaccination or HIV-1 viral load and levels of A(H1N1)pdm09 antibodies. Assessing AID expression before vaccination may be an additional useful tool for defining a vaccination strategy in immune-compromised individuals at risk of immunization failure.

Objective Decreased memory B-cell maintenance during HIV-1 infection has been associated with the viraemia-induced accumulation of activated memory B cells, sensitive to Fas-mediated apoptosis. We aimed at clarifying whether other B-cell subsets might also be affected by an increased Fas expression in HIV-1-infected patients, and we studied the possible contribution of viraemia, lymphopenia or T-cell activation in Fas upregulation on B cells. We analysed whether Fas upregulation might have collaborative effects with the dysregulation of other B-cell modulatory molecules, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) and programmed cell death protein 1 (PD-1), on B-cell homeostasis. Design Fas, LAIR1 and PD-1 were analysed on B-cell subpopulations in HIV-1-infected patients who were treatment naive, nonlymphopenic; antiretroviral therapy (ART)-treated, nonlymphopenic; or ART-treated, lymphopenic or in noninfected controls. Methods Flow cytometry was used to study B-cell subsets and Milliplex for serum cytokines. Results Fas expression increased on all B-cell subpopulations of viraemic or lymphopenic individuals. The decreased ratio of resting memory B cells and their increased Fas expression were not normalized by ART. Cytokines associated with T-cell activation might influence Fas expression on the naive and transitional B cells. LAIR1 expression decreased in all HIV-1-infected patients, but only on memory B cells, whereas PD-1 increased on resting memory B cells in viraemic patients. Conclusion Fas is regulated by the concerted action of viraemia, lymphopenia and T-cell activation during HIV-1 infection, and Fas expression is altered on all peripheral B-cell subsets. Resting memory B-cell homeostasis shows the highest sensitivity to HIV-1-induced perturbations.

Interleukin-7 (IL-7) concentrations are increased in the blood of CD4+ T cell depleted individuals, including HIV-1 infected patients. High IL-7 levels might stimulate T cell activation and, as we have shown earlier, IL-7 can prime resting T cell to CD95 induced apoptosis as well. HIV-1 infection leads to B cell abnormalities including increased apoptosis via the CD95 (Fas) death receptor pathway and loss of memory B cells. Peripheral B cells are not sensitive for IL-7, due to the lack of IL-7Ra expression on their surface; however, here we demonstrate that high IL-7 concentration can prime resting B cells to CD95-mediated apoptosis via an indirect mechanism. T cells cultured with IL-7 induced high CD95 expression on resting B cells together with an increased sensitivity to CD95 mediated apoptosis. As the mediator molecule responsible for B cell priming to CD95 mediated apoptosis we identified the cytokine IFN-γ that T cells secreted in high amounts in response to IL-7. These results suggest that the lymphopenia induced cytokine IL-7 can contribute to the increased B cell apoptosis observed in HIV-1 infected individuals.

T-cell depletion associated with HIV infection or cytoreductive therapies triggers potential T-cell regenerative mechanisms such as peripheral T-lymphocyte expansion to weak antigenic stimuli and the increased availability of interleukin-7 (IL-7), a cytokine with potent antiapoptotic and proliferative activities. Deleterious mechanisms also associated with lymphopenia, such as increased Fas expression and apoptosis of T cell, however, may result in opposing effects. In this study, we show that Fas molecules, primarily associated with T-cell depletion in lymphopenic settings, may also contribute to compensatory T-cell expansion through transmitting costimulatory signals to suboptimally activated T cells. Proliferation of T lymphocytes in response to concomitant Fas and T-cell receptor (TCR) triggering was shown to be increased in HIV-infected individuals compared with noninfected controls. As IL-7 levels are often elevated in lymphopenic individuals in association with increased Fas expression, we analyzed whether IL-7 would influence Fas-mediated proliferative signals in T cells. We show that IL-7 is able to increase the efficacy of Fas to induce proliferation of suboptimally activated T cells. Thus, high IL-7 levels associated with lymphopenic conditions may simultaneously induce sensitivity to Fas-mediated apoptosis in nonactivated T cells and increase Fas-induced costimulatory signals in T cells recognizing low-affinity antigens.

Human natural killer (NK) (CD3- CD56+) cells can be divided into two functionally distinct subsets, CD3- CD56(dim) and CD3- CD56(bright). We analysed the distribution of NK cell subsets in primary and chronic human immunodeficiency virus-1 (HIV-1) infection, to determine if HIV infection stage may influence the subset distribution. In primary infection, contrary to chronic infection, the CD3- CD56(dim) subset was expanded compared to healthy controls. We also studied the effect of antiretroviral therapy administered early in infection and found that NK cell subset distribution was partially restored after 6 months of antiretroviral therapy in primary infection, but not normalized. Recently, NK cells have been divided into CD27- and CD27+ subsets with different migratory and functional capacity and CD27-mediated NK cell activation has been described in mice. We therefore investigated whether CD27 and/or CD70 (CD27 ligand) expression on NK cells, and thus the distribution of these novel NK subsets, was altered in HIV-1-infected patients. We found up-regulated expression of both CD27 and CD70 on NK cells of patients, resulting in higher proportions of CD27(high) and CD70(high) NK cells, and this phenomenon was more pronounced in chronic infection. Experiments conducted in vitro suggest that the high interleukin-7 levels found during HIV-1 infection may participate in up-regulation of CD70 on NK cell subsets. Imbalance of NK cell subsets and up-regulated expression of CD27 and CD70 initiated early in HIV-1 infection may indicate NK cell activation and intrinsic defects initiated by HIV-1 to disarm the innate immune response to the virus.