Polyclonal expansion of cervical cytobrush‐derived T cells to investigate HIV‐specific responses in the female genital tract

Human immunodeficiency virus (HIV) ‐specific T‐cell responses are detectable in the female genital tract of HIV‐infected women but little is known about their frequency or the factors that influence their detection. We investigated the feasibility of polyclonal in vitro expansion of cervical cytobrush‐derived T cells to investigate HIV‐specific responses in the female genital tract in HIV‐infected women. Cytobrush‐derived cervical cells were isolated from 22 HIV‐infected women and expanded with anti‐CD3 and recombinant interleukin‐2. Cervical T‐cell lines were investigated for Gag‐specific responses by interferon‐γ ELISPOT and compared with those detected in matched blood samples. Cervical T‐cell lines were established from 16/22 (72·7%) participants. Although the absolute number of CD3^± cells recovered after expansion was positively associated with the number of cells isolated ex vivo (P = 0·01; R = 0·62), we observed a significant negative correlation between fold expansion and ex vivo cell number (P = 0·004; R = ?0·68). We show that both the magnitude (P = 0·002; R = 0·7) and specific Gag regions targeted by cervical T‐cell lines (P < 0·0001; R = 0·5) correlated significantly with those detected in blood. With one exception, cervical interferon‐γ T‐cell responses to Gag were detected only in HIV‐infected women with blood Gag‐specific response > 1000 spot‐forming units/10⁶ cells. We conclude that cervical Gag‐specific T‐cell responses in expanded lines are most easily detectable in women who have corresponding high‐magnitude Gag‐specific T‐cell responses in blood.     

NYVAC immunization induces polyfunctional HIV‐specific T‐cell responses in chronically‐infected,ART‐treated HIV patients

We report the results of the Theravac‐01 phase I trial, which was conducted to evaluate the safety and immunogenicity of a poxvirus‐based vector, NYVAC, expressing Gag, Pol, Nef, and Env from an HIV clade B isolate. NYVAC‐B vaccine was injected intra‐muscularly into ten HIV‐infected patients successfully treated with antiretroviral therapy, twice on day 0 and again at week 4. Safety and immunogenicity were monitored for 48 weeks. HIV‐specific T‐cell responses following immunization were quantitatively analyzed using an IFN‐γ ELISPOT assay and qualitatively characterized for their functional profile (including multiple cytokines secretion plus cytotoxic and proliferation capacity) by polychromatic flow cytometry. Our results indicate that the NYVAC‐B vaccine is safe and highly immunogenic, as indicated by increased HIV‐specific T‐cell responses in virtually all vaccinees. Interestingly, both an expansion of preexisting T‐cell responses, and the appearance of newly detected HIV‐specific CD4⁺ and CD8⁺ T‐cell responses were observed. Furthermore, immunization mostly induced an increase in Gag‐specific T‐cell responses. In conclusion, NYVAC‐B immunization induces broad, vigorous, and polyfunctional HIV‐specific T‐cell responses, suggesting that poxvirus‐based vaccine regimens may be instrumental in the therapeutic HIV vaccine field.     

Interleukin‐10‐secreting T cells define a suppressive subset within the HIV‐1‐specific T‐cell population

Recent studies have indicated that Treg contribute to the HIV type 1 (HIV‐1)‐related immune pathogenesis. However, it is not clear whether T cells with suppressive properties reside within the HIV‐1‐specific T‐cell population. Here, PBMC from HIV‐1‐infected individuals were stimulated with a 15‐mer Gag peptide pool, and HIV‐1‐specific T cells were enriched by virtue of their secretion of IL‐10 or IFN‐γ using immunomagnetic cell‐sorting. Neither the IL‐10‐secreting cells nor the IFN‐γ‐secreting cells expressed the Treg marker FOXP3, yet the IL‐10‐secreting cells potently suppressed anti‐CD3/CD28‐induced CD4⁺ as well as CD8⁺ T‐cell proliferative responses. As shown by intracellular cytokine staining, IL‐10‐ and IFN‐γ‐producing T cells represent distinct subsets of the HIV‐1‐specific T cells. Our data collectively suggest that functionally defined HIV‐1‐specific T‐cell subsets harbor potent immunoregulatory properties that may contribute to HIV‐1‐associated T‐cell dysfunction.     

HIV gag‐specific immune response mediated by double negative (CD3+CD4−CD8−) T cells in HIV‐exposed seronegative individuals

Double negative (DN) T cells are CD3⁺, CD4^?, CD8^? cells with either T‐cell receptors (TCR) αβ or TCR γδ whose importance on protection against HIV infection is unknown. Since HIV‐exposed seronegative individuals correspond to an ideal group in whom correlates of protection are expected, the role of these cells was studied in 13 HIV‐serodiscordant couples in a stable relationship and reporting unprotected sexual intercourses. HIV‐specific immune responses mediated by DN T‐cells were evaluated by measuring intracellular IFNγ and MIP1β (CCL4) production in response to HIV‐Gag peptides. Thirty‐five healthy controls not exposed to HIV were tested similarly and used to define a threshold for positive responses. Interestingly, Gag‐specific DN T‐cell responses were found in 3/13 (23%) HIV‐exposed seronegative individuals (Group A), involving both DN/αβ⁺ and DN/γδ⁺ T‐cells through MIP1β and IFNγ production. 4/13 (30%) of partners infected with HIV (Group B) also showed Gag‐specific responses but were mediated exclusively by DN/γδ⁺ T‐cells, mainly through IFNγ production. DN T‐cells in Group A individuals can display differential HIV‐specific immune responses, which might contribute to the low susceptibility to infection with HIV shown by individuals in Group A. J. Med. Virol. 85:200–209, 2013. © 2012 Wiley Periodicals, Inc.     

Novel HIV‐1 clade B candidate vaccines designed for HLA‐B*5101+ patients protected mice against chimaeric ecotropic HIV‐1 challenge

Novel candidate HIV‐1 vaccines have been constructed, which are tailor‐designed for HLA‐B^*5101⁺ patients infected with HIV‐1 clade B. These vaccines employ novel immunogen HIVB‐B^*5101 derived from consensus HIV‐1 clade B Gag p17 and p24 regions coupled to two Pol‐derived B^*5101‐restricted epitopes, which are together with a third B^*5101 epitope in Gag dominant in HIV‐1‐infected long‐term non‐progressing patients. Both plasmid DNA and modified vaccinia virus Ankara (MVA) vectors supported high expression levels of the HIVB‐B^*5101 immunogen in cultured cells. Heterologous DNA prime‐recombinant MVA boost regimen induced efficiently HIV‐1‐specific CD8⁺ T‐cell responses in BALB/c mice. These vaccine‐elicited T cells were multifunctional, killed efficiently target cells in vivo, and protected mice against challenge with ecotropic HIV‐1/NL4‐3 and ecotropic HIV‐1/NDK chimaeric viruses with HIV‐1 clade B or D backbones, respectively, and ecotropic murine leukemia virus gp80 envelope, and therefore did so in the absence of anti‐HIV‐1 gp120 antibodies. These results support further development of HIVB‐B^*5101 vaccines in combined heterologous‐modality regimens. The use of allele‐specific vaccines in humans is discussed in the context of other developments in the HIV‐1 field.     

Human leucocyte antigen‐Bw4 and Gag‐specific T cell responses are associated with slow disease progression in HIV‐1B‐infected anti‐retroviral therapy‐naive Chinese

In China, the majority of human immunodeficiency virus (HIV) infections are predominately subtype B. It is important to characterize the HIV‐1 subtype B‐specific and its T cell response within the Chinese population, with the aim of identifying protective correlates of immunity to control HIV‐1 infections. In this study, we performed a comprehensive analysis looking into the magnitude/strength of T cell responses directed at the Gag protein of the HIV‐1 subtype B, one of the most conserved HIV‐1 proteins. The study group consisted of anti‐retroviral native and chronic HIV‐1 subtype B‐infected individuals. We used enzyme‐linked immunospot (ELISPOT) assay to quantify the total T cell responses to HIV‐1 Gag at the single peptide level. Twenty‐eight (38%) peptides were recognized in 24 (82·8%) individuals. The p24 was identified as the most frequently recognized subunit protein with the greatest T cell response in the test, which correlated positively with CD4⁺ T cell count and inversely with viral load (VL). At the level of the human leucocyte antigen (HLA) supertypes, we detected the highest levels and a significant correlation with both the CD4⁺ T cell count and the VL with Gag T cell responses in Bw4/Bw4. These findings demonstrate that (i) the HIV‐1B Gag p24‐specific immune responses play an important role in controlling viral replication and slowing clinical progression; and (ii) HLA‐Bw4/Bw4 allele has stronger T cell responses, which is associated with slow clinical progression in Chinese HIV patients.     

Emergence of a distinct HIV‐specific IL‐10‐producing CD8+ T‐cell subset with immunomodulatory functions during chronic HIV‐1 infection

Interleukin‐10 (IL‐10) plays a key role in regulating proinflammatory immune responses to infection but can interfere with pathogen clearance. Although IL‐10 is upregulated throughout HIV‐1 infection in multiple cell subsets, whether this is a viral immune evasion strategy or an appropriate response to immune activation is unresolved. Analysis of IL‐10 production at the single cell level in 51 chronically infected subjects (31 antiretroviral (ART) naïve and 20 ART treated) showed that a subset of CD8⁺ T cells with a CD25^neg FoxP3^neg phenotype contributes substantially to IL‐10 production in response to HIV‐1 gag stimulation. The frequencies of gag‐specific IL‐10‐ and IFN‐γ‐producing T cells in ART‐naïve subjects were strongly correlated and the majority of these IL‐10⁺ CD8⁺ T cells co‐produced IFN‐γ; however, patients with a predominant IL‐10⁺/IFN‐γ^neg profile showed better control of viraemia. Depletion of HIV‐specific CD8⁺ IL‐10⁺ cells from PBMCs led to upregulation of CD38 on CD14⁺ monocytes together with increased IL‐6 production, in response to gag stimulation. Increased CD38 expression was positively correlated with the frequency of the IL‐10⁺ population and was also induced by exposure of monocytes to HIV‐1 in vitro. Production of IL‐10 by HIV‐specific CD8⁺ T cells may represent an adaptive regulatory response to monocyte activation during chronic infection.     

Blocking of integrins inhibits HIV‐1 infection of human cervical mucosa immune cells with free and complement‐opsonized virions

The initial interaction between HIV‐1 and the host occurs at the mucosa during sexual intercourse. In cervical mucosa, HIV‐1 exists both as free and opsonized virions and this might influence initial infection. We used cervical explants to study HIV‐1 transmission, the effects of opsonization on infectivity, and how infection can be prevented. Complement opsonization enhanced HIV‐1 infection of dendritic cells (DCs) compared with that by free HIV‐1, but this increased infection was not observed with CD4⁺ T cells. Blockage of the α4‐, β7‐, and β1‐integrins significantly inhibited HIV‐1 infection of both DCs and CD4⁺ T cells. We found a greater impairment of HIV‐1 infection in DCs for complement‐opsonized virions compared with that of free virions when αM/β2‐ and α4‐integrins were blocked. Blocking the C‐type lectin receptor macrophage mannose receptor (MMR) inhibited infection of emigrating DCs but had no effect on CD4⁺ T‐cell infection. We show that blocking of integrins decreases the HIV‐1 infection of both mucosal DCs and CD4⁺ T cells emigrating from the cervical tissues. These findings may provide the basis of novel microbicidal strategies that may help limit or prevent initial infection of the cervical mucosa, thereby reducing or averting systemic HIV‐1 infection.     

Highly avid,oligoclonal, early‐differentiated antigen‐specific CD8+ T cells in chronic HIV‐2 infection

HIV‐1‐specific CD8⁺ T cells are present in most HIV‐1‐infected people and play an important role in controlling viral replication, but the characteristics of an effective HIV‐specific T‐cell response are largely unknown. The majority of HIV‐2‐infected people behave as long‐term non‐progressors while those who progress to AIDS do so in a manner indistinguishable from HIV‐1. A detailed study of HIV‐2 infection may identify protective immune responses. Robust gag p26‐specific T‐cell responses are elicited during HIV‐2 infection and correlate with control of viremia. In this study, we analyzed features of an HLA‐B^*3501‐restricted T‐cell response to HIV‐2 p26 that may contribute to virus control. In contrast to HIV‐1, HIV‐2‐specific T cells are at an early stage of differentiation (CD27⁺CD28⁺), a finding that relates directly to CD4⁺ T‐cell levels and inversely to immune activation. The cells demonstrate IFN‐γ secretion, oligoclonal T‐cell receptor Vβ gene segment usage, exceptional avidity and secretion of pro‐inflammatory cytokines. Despite the potentially strong selection pressure imposed on the virus by these cells, there was no evidence of HIV‐2 sequence evolution. We propose that in chronic HIV‐2 infection, the maintenance of early‐differentiated, highly avid CD8⁺ T cells could account for the non‐progressive course of disease. Such responses may be desirable from an HIV vaccine.     

Comprehensive analysis of HIV Gag‐specific IFN‐γ response in HIV‐1‐ and HIV‐2‐infected asymptomatic patients from a clinical cohort in The Gambia

Majority of HIV‐2‐infected individuals meet the criteria of long‐term non‐progressors. This has been linked to superior qualitative HIV‐2‐specific cellular immune responses that correlate with viral control. However, it is unknown whether this is due to frequent targeting of immunodominant Gag epitopes in HIV‐2 than HIV‐1 infection. We describe a comprehensive comparison of the magnitude, breadth and frequency of Gag responses and the degree of cross‐recognition of frequently targeted, immunodominant Gag peptides in a cross‐sectional study of asymptomatic HIV‐1‐ and HIV‐2‐infected individuals. Fresh PBMC from 20 HIV‐1‐ and 20 HIV‐2‐infected patients with similar CD4⁺ T‐cell counts (p=0.36) were stimulated with pools of HIV‐1 and/or HIV‐2 Gag peptides in an IFN‐γ ELISPOT assay. We found no difference in the cumulative magnitude of IFN‐γ responses (p=0.75) despite significantly lower plasma viral loads in HIV‐2‐infected people (p<0.0001). However, Gag211–290 was targeted with significantly higher magnitude in HIV‐2‐infected subjects (p=0.03) although this did not correlate with viral control. There was no difference in frequently targeted Gag peptides, the breadth, immunodominance or cross‐recognition of Gag peptide pools between the two infections. This suggests that other factors may control viral replication in HIV‐2 infection.     

Loss of DNAM‐1 contributes to CD8+ T‐cell exhaustion in chronic HIV‐1 infection

The hallmark of chronic viral infections is a progressive exhaustion of antigen‐specific CD8⁺ T cells that leads to persisting viral replication. It is generally believed that exhaustion is a consequence of the accumulation of multiple inhibitory receptors on CD8⁺ T cells that makes them dysfunctional. Here, we show that during human chronic HIV‐1 infection, a CD8⁺ T‐cell positive costimulatory pathway mediated by DNAX‐activating molecule‐1 is also disrupted. Thus, DNAX‐activating molecule‐1 downregulation on CD8⁺ T cells aggravates the impairment of CTL effector function in chronic HIV‐1 infection.     

CTL recognition of HIV‐1‐infected cells via cross‐recognition of multiple overlapping peptides from a single 11‐mer Pol sequence

It is known that overlapping HIV‐1 peptides of different lengths can be presented by a given HLA class I molecule. However, the role of those peptides in CD8⁺ T cells recognition of HIV‐1‐infected cells remains unclear. Here we investigated the recognition of overlapping 8‐mer to 11‐mer peptides of Pol 155–165 by HLA‐B*54:01‐restricted CD8⁺ T cells. The analysis of ex vivo T cells using ELISPOT and tetramer binding assays showed that there were different patterns of CD8⁺ T‐cell responses to these peptides among chronically HIV‐1‐infected HLA‐B*54:01⁺ individuals, though the response to the 9‐mer peptide was the strongest among them. CD8⁺ T‐cell clones with TCRs specific for the 9‐mer, 10‐mer, and/or 11‐mer peptides effectively killed HIV‐1‐infected cells. Together, these results suggest that the 9‐mer and 10‐mer peptides could be predominantly presented by HLA‐B*54:01, though it remains possible that the 11‐mer peptide was also presented by this HLA allele. The present study demonstrates effective CD8⁺ T‐cell recognition of HIV‐1‐infected cells via presentation of multiple overlapping HIV‐1 peptides and cross‐recognition by the CD8⁺ T cells.     

HIV–TB coinfection impairs CD8+ T‐cell differentiation and function while dehydroepiandrosterone improves cytotoxic antitubercular immune responses

Tuberculosis (TB) is the leading cause of death among HIV‐positive patients. The decreasing frequencies of terminal effector (T_TE) CD8⁺T cells may increase reactivation risk in persons latently infected with Mycobacterium tuberculosis (Mtb). We have previously shown that dehydroepiandrosterone (DHEA) increases the protective antitubercular immune responses in HIV–TB patients. Here, we aimed to study Mtb‐specific cytotoxicity, IFN‐γ secretion, memory status of CD8⁺T cells, and their modulation by DHEA during HIV–TB coinfection. CD8⁺T cells from HIV–TB patients showed a more differentiated phenotype with diminished naïve and higher effector memory and T_TE T‐cell frequencies compared to healthy donors both in total and Mtb‐specific CD8⁺T cells. Notably, CD8⁺T cells from HIV–TB patients displayed higher Terminal Effector (T_TE) CD45RA^dim proportions with lower CD45RA expression levels, suggesting a not fully differentiated phenotype. Also, PD‐1 expression levels on CD8⁺T cells from HIV–TB patients increased although restricted to the CD27⁺ population. Interestingly, DHEA plasma levels positively correlated with T_TE in CD8⁺T cells and in vitro DHEA treatment enhanced Mtb‐specific cytotoxic responses and terminal differentiation in CD8⁺T cells from HIV–TB patients. Our data suggest that HIV–TB coinfection promotes a deficient CD8⁺ T‐cell differentiation, whereas DHEA may contribute to improving antitubercular immunity by enhancing CD8⁺T‐cell functions during HIV–TB coinfection.     

Partial recovery of senescence and differentiation disturbances in CD8+ T cell effector‐memory cells in HIV‐1 infection after initiation of anti‐retroviral treatment

Immune senescence as well as disturbed CD8⁺ T cell differentiation are a hallmark of chronic HIV infection. Here, we investigated to what extent immune senescence is reversible after initiation of anti‐retroviral treatment (ART). Peripheral blood mononuclear cells (PBMCs) from a cohort of HIV patients with different disease courses, including untreated viral controllers (n = 10), viral non‐controllers (n = 16) and patients on ART (n = 20), were analysed and compared to uninfected controls (n = 25) by flow cytometry on bulk and HIV‐specific major histocompatibility complex (MHC) class I tetramer⁺ CD8⁺ T cells for expression of the memory markers CCR7 and CD45RO, as well as the senescence marker CD57 and the differentiation and survival marker CD127. Furthermore, a subset of patients was analysed longitudinally before and after initiation of ART. Frequencies of CD57⁺CD8⁺ T cells decreased after initiation of ART in central memory (Tcm) but not in effector memory T cell populations (TemRO and TemRA). The frequency of CD127⁺CD8⁺ cells increased in Tcm and TemRO. We observed a reduction of CD127^– T cells in Tcm, TemRO and partially in TemRA subsets after initiation of ART. Importantly, HIV‐specific CD8⁺ TemRO cells predominantly displayed a CD127^–CD57⁺ phenotype in untreated HIV‐patients, whereas the CD127⁺CD57^– phenotype was under‐represented in these patients. The frequency of the CD127⁺CD57^–CD8⁺ T cell subpopulation correlated strongly with absolute CD4⁺ counts in HIV‐infected patients before and after initiation of ART. These findings can be interpreted as a phenotypical correlate of CD8⁺ memory T cell differentiation and the premature ‘ageing’ of the immune system, which was even observed in successfully virally suppressed HIV patients.     

Plasmacytoid dendritic cell and functional HIV Gag p55‐specific T cells before treatment interruption can inform set‐point plasma HIV viral load after treatment interruption in chronically suppressed HIV‐1+ patients

The identification of immune correlates of HIV control is important for the design of immunotherapies that could support cure or antiretroviral therapy (ART) intensification-related strategies. ART interruptions may facilitate this task through exposure of an ART partially reconstituted immune system to endogenous virus. We investigated the relationship between set-point plasma HIV viral load (VL) during an ART interruption and innate/adaptive parameters before or after interruption. Dendritic cell (DC), natural killer (NK) cell and HIV Gag p55-specific T-cell functional responses were measured in paired cryopreserved peripheral blood mononuclear cells obtained at the beginning (on ART) and at set-point of an open-ended interruption from 31 ART-suppressed chronically HIV-1⁺ patients. Spearman correlation and linear regression modeling were used. Frequencies of plasmacytoid DC (pDC), and HIV Gag p55-specific CD3⁺ CD4⁻ perforin⁺ IFN-γ⁺ cells at the beginning of interruption associated negatively with set-point plasma VL. Inclusion of both variables with interaction into a model resulted in the best fit (adjusted R² = 0·6874). Frequencies of pDC or HIV Gag p55-specific CD3⁺ CD4⁻ CSFE^lo CD107a⁺ cells at set-point associated negatively with set-point plasma VL. The dual contribution of pDC and anti-HIV T-cell responses to viral control, supported by our models, suggests that these variables may serve as immune correlates of viral control and could be integrated in cure or ART-intensification strategies.     

Interleukin‐18 activates Vγ9Vδ2+ T cells from HIV‐positive individuals: recovering the response to phosphoantigen

The study aimed to identify an immunoregulatory factor that restores the phosphoantigen response of Vγ9Vδ2⁺ T cells from HIV‐positive individuals on antiretroviral therapy. It was designed to characterize the effects of interleukin‐18 (IL‐18) on proliferation and effector function in Vγ9Vδ2 T cells from HIV‐negative individuals and test whether exogenous IL‐18 reconstitutes the Vγ9Vδ2 T‐cell response to phosphoantigen from HIV‐positive donors. Vγ9Vδ2 T cells from HIV‐negative individuals responded strongly to phosphoantigen or aminobisphosphonate stimulation of peripheral blood mononuclear cells (PBMC), whereas cells with similar T‐cell receptor profiles from HIV‐positive individuals only responded to aminobisphosphonate. Interleukin‐18 was higher after aminobisphosphonate stimulation due to activation of the inflammasome pathway. Both IL‐18 and IL‐18 receptor levels were measured and the activity of exogenous IL‐18 on HIV‐negative and HIV‐positive PBMC was evaluated in terms of Vγ9Vδ2 T‐cell proliferation, memory subsets, cytokine expression and CD107a expression. Interleukin‐18 stimulation increased proliferation, enhanced the accumulation of effector memory cells, and increased expression of cytotoxic markers in HIV‐negative controls. When Vγ9Vδ2 T cells from HIV‐positive individuals were stimulated with isopentenyl pyrophosphate in the presence of IL‐18, there was increased proliferation, accumulation of memory cells, and higher expression of CD56, NKG2D and CD107a (markers of cytotoxic effector phenotype). Interleukin‐18 stimulation specifically expanded the Vγ9‐JγP⁺ subset of Vγ9Vδ2 T cells, as was expected for normal responses to phosphoantigen. Interleukin‐18 is a potent stimulator of Vγ9Vδ2 T‐cell proliferation and effector function. Therapies directed at reconstituting Vγ9Vδ2 T‐cell activity in HIV‐positive individuals should include stimulators of IL‐18 or direct cytokine supplementation.     

Adenovirus 5‐ and 35‐based immunotherapy enhances the strength but not breadth or quality of immunity during chronic SIV infection

Heterologous adenovirus‐based vectors hold promise as preventative HIV vaccines but their capacity to induce effective T‐cell immunity in established infection has not been explored. We vaccinated rhesus macaques chronically infected with SIVmac251 and undergoing antiretroviral therapy (ART) with human adenovirus serotype 5‐based vectors expressing SIV Gag, Env, and Nef with and without IL‐15 and evaluated vaccine immunogenicity. Vaccination increased Ag‐specific T cells 20‐fold but did not expand the breadth of epitopes recognized or the quality of response, as the majority of CD8⁺ and CD4⁺ T cells produced only one cytokine irrespective of vaccination. Immunization transiently restored blood CD4⁺ central memory T cells (Tcm) and boosted CD4⁺ and CD8⁺ Tcm and effector cell responses but did not prevent virus rebound upon cessation of ART. Boosting with human adenovirus serotype 35‐based vectors during a second ART cycle increased Ag‐specific T cells to 50‐fold above pre‐vaccination levels and boosted CD4⁺ Tcm numbers but did not expand the breadth or quality of immunity or control virus levels following drug discontinuation. The number of blood CD4⁺ Tcm correlated positively with complexity of T‐cell responses and negatively with virus load, suggesting that more complete restoration of this subset through vaccination would be beneficial.     

Naturally processed HLA‐DR3‐restricted HHV‐6B peptides are recognized broadly with polyfunctional and cytotoxic CD4 T‐cell responses

Human herpes virus 6B (HHV‐6B) is a widespread virus that infects most people early in infancy and establishes a chronic life‐long infection with periodic reactivation. CD4 T cells have been implicated in control of HHV‐6B, but antigenic targets and functional characteristics of the CD4 T‐cell response are poorly understood. We identified 25 naturally processed MHC‐II peptides, derived from six different HHV‐6B proteins, and showed that they were recognized by CD4 T‐cell responses in HLA‐matched donors. The peptides were identified by mass spectrometry after elution from HLA‐DR molecules isolated from HHV‐6B‐infected T cells. The peptides showed strong binding to matched HLA alleles and elicited recall T‐cell responses in vitro. T‐cell lines expanded in vitro were used for functional characterization of the response. Responding cells were mainly CD3⁺CD4⁺, produced IFN‐γ, TNF‐α, and low levels of IL‐2, alone or in combination, highlighting the presence of polyfunctional T cells in the overall response. Many of the responding cells mobilized CD107a, stored granzyme B, and mediated specific killing of peptide‐pulsed target cells. These results highlight a potential role for polyfunctional cytotoxic CD4 T cells in the long‐term control of HHV‐6B infection.     

Human antigen‐specific CD4+CD25+CD134+CD39+ T cells are enriched for regulatory T cells and comprise a substantial proportion of recall responses

Human Ag‐specific CD4⁺ T cells can be detected by their dual expression of CD134 (OX40) and CD25 after a 44 hours stimulation with cognate Ag. We show that surface expression of CD39 on Ag‐specific cells consistently identifies a substantial population of CD4⁺CD25⁺CD134⁺CD39⁺ T cells that have a Treg‐cell‐like phenotype and mostly originate from bulk memory CD4⁺CD45RO⁺CD127^lowCD25^highCD39⁺ Treg cells. Viable, Ag‐specific CD25⁺CD134⁺CD39⁺ T cells could be expanded in vitro as cell lines and clones, and retained high Forkhead Box Protein 3, CTLA‐4 and CD39 expression, suppressive activity and Ag specificity. We also utilised this combination of cell surface markers to measure HIV‐Gag responses in HIV⁺ patients before and after anti‐retroviral therapy (ART). Interestingly, we found that the percentage of CD39^? cells within baseline CD4⁺ T‐cell responses to HIV‐Gag was negatively correlated with HIV viral load pre‐ART and positively correlated with CD4⁺ T‐cell recovery over 96 weeks of ART. Collectively, our data show that Ag‐specific CD4⁺CD25⁺CD134⁺CD39⁺ T cells are highly enriched for Treg cells, form a large component of recall responses and maintain a Treg‐cell‐like phenotype upon in vitro expansion. Identification and isolation of these cells enables the role of Treg cells in memory responses to be further defined and provides a development pathway for novel therapeutics.     

NK cells are primed by ANRS MVAHIV‐infected DCs,via a mechanism involving NKG2D and membrane‐bound IL‐15, to control HIV‐1 infection in CD4+ T cells

Natural killer (NK) cells are the major antiviral effector cell population of the innate immune system. It has been demonstrated that NK‐cell activity can be modulated by the interaction with dendritic cells (DCs). The HIV‐1 vaccine candidate Modified Vaccinia Ankara encoding an HIV polypeptide (MVA_HIV), developed by the French National Agency for Research on AIDS (ANRS), has the ability to prime NK cells to control HIV‐1 infection in DCs. However, whether or not MVA_HIV‐primed NK cells are able to better control HIV‐1 infection in CD4⁺ T cells, and the mechanism underlying the specific priming, remain undetermined. In this study, we show that MVA_HIV‐primed NK cells display a greater capacity to control HIV‐1 infection in autologous CD4⁺ T cells. We also highlight the importance of NKG2D engagement on NK cells and DC‐produced IL‐15 to achieve the anti‐HIV‐1 specific priming, as blockade of either NKG2D or IL‐15 during MVA_HIV‐priming lead to a subsequent decreased control of HIV‐1 infection in autologous CD4⁺ T cells. Furthermore, we show that the decreased control of HIV‐1 infection in CD4⁺ T cells might be due, at least in part, to the decreased expression of membrane‐bound IL‐15 (mbIL‐15) on DCs when NKG2D is blocked during MVA_HIV‐priming of NK cells.