Additive effects of booster mRNA vaccination and SARS-CoV-2 Omicron infection on T cell immunity across immunocompromised states.

Suboptimal immunity to SARS-CoV-2 mRNA vaccination has frequently been observed in individuals with various immunodeficiencies. Given the increased antibody evasion properties of emerging SARS-CoV-2 subvariants, it is necessary to assess whether other components of adaptive immunity generate resilient and protective responses against infection. We assessed T cell responses in 279 individuals, covering five different immunodeficiencies and healthy controls, before and after booster mRNA vaccination, as well as after Omicron infection in a subset of patients. We observed robust and persistent Omicron-reactive T cell responses that increased markedly upon booster vaccination and correlated directly with antibody titers across all patient groups. Poor vaccination responsiveness in immunocompromised or elderly individuals was effectively counteracted by the administration of additional vaccine doses. Functionally, Omicron-reactive T cell responses exhibited a pronounced cytotoxic profile and signs of longevity, characterized by CD45RA+ effector memory subpopulations with stem cell-like properties and increased proliferative capacity. Regardless of underlying immunodeficiency, booster-vaccinated and Omicron-infected individuals appeared protected against severe disease and exhibited enhanced and diversified T cell responses against conserved and Omicron-specific epitopes. Our findings indicate that T cells retain the ability to generate highly functional responses against newly emerging variants, even after repeated antigen exposure and a robust immunological imprint from ancestral SARS-CoV-2 mRNA vaccination.

Functional SARS-CoV-2 cross-reactive CD4+ T cells established in early childhood decline with age.

Pre-existing SARS-CoV-2-reactive T cells have been identified in SARS-CoV-2-unexposed individuals, potentially modulating COVID-19 and vaccination outcomes. Here, we provide evidence that functional cross-reactive memory CD4+ T cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is established in early childhood, mirroring early seroconversion with seasonal human coronavirus OC43. Humoral and cellular immune responses against OC43 and SARS-CoV-2 were assessed in SARS-CoV-2-unexposed children (paired samples at age two and six) and adults (age 26 to 83). Pre-existing SARS-CoV-2-reactive CD4+ T cell responses targeting spike, nucleocapsid, and membrane were closely linked to the frequency of OC43-specific memory CD4+ T cells in childhood. The functional quality of the cross-reactive memory CD4+ T cell responses targeting SARS-CoV-2 spike, but not nucleocapsid, paralleled OC43-specific T cell responses. OC43-specific antibodies were prevalent already at age two. However, they did not increase further with age, contrasting with the antibody magnitudes against HKU1 (ß-coronavirus), 229E and NL63 (α-coronaviruses), rhinovirus, Epstein-Barr virus (EBV), and influenza virus, which increased after age two. The quality of the memory CD4+ T cell responses peaked at age six and subsequently declined with age, with diminished expression of interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF), and CD38 in late adulthood. Age-dependent qualitative differences in the pre-existing SARS-CoV-2-reactive T cell responses may reflect the ability of the host to control coronavirus infections and respond to vaccination.

Hierarchical Clustering and Trajectory Analyses Reveal Viremia-Independent B-Cell Perturbations in HIV-2 Infection.

Time to AIDS in HIV-2 infection is approximately twice as long compared to in HIV-1 infection. Despite reduced viremia, HIV-2-infected individuals display signs of chronic immune activation. In HIV-1-infected individuals, B-cell hyperactivation is driven by continuous antigen exposure. However, the contribution of viremia to B-cell perturbations in HIV-2-infected individuals remains largely unexplored. Here, we used polychromatic flow cytometry, consensus hierarchical clustering and pseudotime trajectory inference to characterize B-cells in HIV-1- or HIV-2-infected and in HIV seronegative individuals. We observed increased frequencies of clusters containing hyperactivated T-bethighCD95highCD27int and proliferating T-bet+CD95highCD27+CD71+ memory B-cells in viremic HIV-1 (p < 0.001 and p < 0.001, respectively), viremic HIV-2 (p < 0.001 and p = 0.014, respectively) and in treatment-naïve aviremic HIV-2 (p = 0.004 and p = 0.020, respectively)-infected individuals, compared to seronegative individuals. In contrast, these expansions were not observed in successfully treated HIV-1-infected individuals. Finally, pseudotime trajectory inference showed that T-bet-expressing hyperactivated and proliferating memory B-cell populations were located at the terminal end of two trajectories, in both HIV-1 and HIV-2 infections. As the treatment-naïve aviremic HIV-2-infected individuals, but not the successfully ART-treated HIV-1-infected individuals, showed B-cell perturbations, our data suggest that aviremic HIV-2-infected individuals would also benefit from antiretroviral treatment.

Ancestral SARS-CoV-2-specific T cells cross-recognize the Omicron variant.

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron (B.1.1.529) variant of concern (VOC) has destabilized global efforts to control the impact of coronavirus disease 2019 (COVID-19). Recent data have suggested that B.1.1.529 can readily infect people with naturally acquired or vaccine-induced immunity, facilitated in some cases by viral escape from antibodies that neutralize ancestral SARS-CoV-2. However, severe disease appears to be relatively uncommon in such individuals, highlighting a potential role for other components of the adaptive immune system. We report here that SARS-CoV-2 spike-specific CD4+ and CD8+ T cells induced by prior infection or BNT162b2 vaccination provide extensive immune coverage against B.1.1.529. The median relative frequencies of SARS-CoV-2 spike-specific CD4+ T cells that cross-recognized B.1.1.529 in previously infected or BNT162b2-vaccinated individuals were 84% and 91%, respectively, and the corresponding median relative frequencies for SARS-CoV-2 spike-specific CD8+ T cells were 70% and 92%, respectively. Pairwise comparisons across groups further revealed that SARS-CoV-2 spike-reactive CD4+ and CD8+ T cells were functionally and phenotypically similar in response to the ancestral strain or B.1.1.529. Collectively, our data indicate that established SARS-CoV-2 spike-specific CD4+ and CD8+ T cell responses, especially after BNT162b2 vaccination, remain largely intact against B.1.1.529.

Inverted CD8 T-Cell Exhaustion and Co-Stimulation Marker Balance Differentiate Aviremic HIV-2-Infected From Seronegative Individuals.

HIV-2 is less pathogenic compared to HIV-1. Still, disease progression may develop in aviremic HIV-2 infection, but the driving forces and mechanisms behind such development are unclear. Here, we aimed to reveal the immunophenotypic pattern associated with CD8 T-cell pathology in HIV-2 infection, in relation to viremia and markers of disease progression. The relationships between pathological differences of the CD8 T-cell memory population and viremia were analyzed in blood samples obtained from an occupational cohort in Guinea-Bissau, including HIV-2 viremic and aviremic individuals. For comparison, samples from HIV-1- or dually HIV-1/2-infected and seronegative individuals were obtained from the same cohort. CD8 T-cell exhaustion was evaluated by the combined expression patterns of activation, stimulatory and inhibitory immune checkpoint markers analyzed using multicolor flow cytometry and advanced bioinformatics. Unsupervised multidimensional clustering analysis identified a cluster of late differentiated CD8 T-cells expressing activation (CD38+, HLA-DRint/high), co-stimulatory (CD226+/-), and immune inhibitory (2B4+, PD-1high, TIGIThigh) markers that distinguished aviremic from viremic HIV-2, and treated from untreated HIV-1-infected individuals. This CD8 T-cell population displayed close correlations to CD4%, viremia, and plasma levels of IP-10, sCD14 and beta-2 microglobulin in HIV-2 infection. Detailed analysis revealed that aviremic HIV-2-infected individuals had higher frequencies of exhausted TIGIT+ CD8 T-cell populations lacking CD226, while reduced percentage of stimulation-receptive TIGIT-CD226+ CD8 T-cells, compared to seronegative individuals. Our results suggest that HIV-2 infection, independent of viremia, skews CD8 T-cells towards exhaustion and reduced co-stimulation readiness. Further knowledge on CD8 T-cell phenotypes might provide help in therapy monitoring and identification of immunotherapy targets.

Identification of resident memory CD8+ T cells with functional specificity for SARS-CoV-2 in unexposed oropharyngeal lymphoid tissue.

Cross-reactive CD4+ T cells that recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more commonly detected in the peripheral blood of unexposed individuals compared with SARS-CoV-2­reactive CD8+ T cells. However, large numbers of memory CD8+ T cells reside in tissues, feasibly harboring localized SARS-CoV-2­specific immune responses. To test this idea, we performed a comprehensive functional and phenotypic analysis of virus-specific T cells in tonsils, a major lymphoid tissue site in the upper respiratory tract, and matched peripheral blood samples obtained from children and adults before the emergence of COVID-19 (coronavirus disease 2019). We found that SARS-CoV-2­specific memory CD4+ T cells could be found at similar frequencies in the tonsils and peripheral blood in unexposed individuals, whereas functional SARS-CoV-2­specific memory CD8+ T cells were almost only detectable in the tonsils. Tonsillar SARS-CoV-2­specific memory CD8+ T cells displayed a follicular homing and tissue-resident memory phenotype, similar to tonsillar Epstein-Barr virus­specific memory CD8+ T cells, but were functionally less potent than other virus-specific memory CD8+ T cell responses. The presence of preexisting tissue-resident memory CD8+ T cells in unexposed individuals could potentially enable rapid sentinel immune responses against SARS-CoV-2.

The known unknowns of T cell immunity to COVID-19.

Tremendous progress has been made in understanding the role of T cell immunity in acute and convalescent COVID-19 infection. Here we shed light on the "known unknowns" of pre-existing and acquired T cell responses in relation to acute and convalescent SARS-CoV-2 infection.

Delayed Expression of PD-1 and TIGIT on HIV-Specific CD8 T Cells in Untreated HLA-B*57:01 Individuals Followed from Early Infection.

While the relationship of protective human leukocyte antigen (HLA) class I alleles and HIV progression is well defined, the interaction of HLA-mediated protection and CD8 T-cell exhaustion is less well characterized. To gain insight into the influence of HLA-B*57:01 on the deterioration of CD8 T-cell responses during HIV infection in the absence of antiretroviral treatment, we compared HLA-B*57:01-restricted HIV-specific CD8 T-cell responses to responses restricted by other HLA class I alleles longitudinally after control of peak viremia. Detailed characterization of polyfunctionality, differentiation phenotypes, transcription factor, and inhibitory receptor expression revealed progression of CD8 T-cell exhaustion over the course of the infection in both patient groups. However, early effects on the phenotype of the total CD8 T-cell population were apparent only in HLA-B*57-negative patients. The HLA-B*57:01-restricted, HIV epitope-specific CD8 T-cell responses showed beneficial functional patterns and significantly lower frequencies of inhibitory receptor expression, i.e., PD-1 and coexpression of PD-1 and TIGIT, within the first year of infection. Coexpression of PD-1 and TIGIT was correlated with clinical markers of disease progression and declining percentages of the T-bethi Eomesdim CD8 T-cell population. In accordance with clinical and immunological deterioration in the HLA-B*57:01 group, the difference in PD-1 and TIGIT receptor expression did not persist to later stages of the disease.IMPORTANCE Given the synergistic nature of TIGIT and PD-1, the coexpression of those inhibitory receptors should be considered when evaluating T-cell pathogenesis, developing immunomodulatory therapies or vaccines for HIV, and when using immunotherapy or vaccination for other causes in HIV-infected patients. HIV-mediated T-cell exhaustion influences the patient´s disease progression, immune system and subsequently non-AIDS complications, and efficacy of vaccinations against other pathogens. Consequently, the possibilities of interfering with exhaustion are numerous. Expanding the use of immunomodulatory therapies to include HIV treatment depends on information about possible targets and their role in the deterioration of the immune system. Furthermore, the rise of immunotherapies against cancer and elevated cancer incidence in HIV-infected patients together increase the need for detailed knowledge of T-cell exhaustion and possible interactions. A broader approach to counteract immune exhaustion to alleviate complications and improve efficacy of other vaccines also promises to increase patients' health and quality of life.

Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease.

CD4+ T cells subsets have a wide range of important helper and regulatory functions in the immune system. Several studies have specifically suggested that circulating effector CD4+ T cells may play a direct role in control of HIV replication through cytolytic activity or autocrine ß-chemokine production. However, it remains unclear whether effector CD4+ T cells expressing cytolytic molecules and ß-chemokines are present within lymph nodes (LNs), a major site of HIV replication. Here, we report that expression of ß-chemokines and cytolytic molecules are enriched within a CD4+ T cell population with high levels of the T-box transcription factors T-bet and eomesodermin (Eomes). This effector population is predominately found in peripheral blood and is limited in LNs regardless of HIV infection or treatment status. As a result, CD4+ T cells generally lack effector functions in LNs, including cytolytic capacity and IFNγ and ß-chemokine expression, even in HIV elite controllers and during acute/early HIV infection. While we do find the presence of degranulating CD4+ T cells in LNs, these cells do not bear functional or transcriptional effector T cell properties and are inherently poor to form stable immunological synapses compared to their peripheral blood counterparts. We demonstrate that CD4+ T cell cytolytic function, phenotype, and programming in the peripheral blood is dissociated from those characteristics found in lymphoid tissues. Together, these data challenge our current models based on blood and suggest spatially and temporally dissociated mechanisms of viral control in lymphoid tissues.

Human Immunodeficiency Virus-Infected Women Have High Numbers of CD103-CD8+ T Cells Residing Close to the Basal Membrane of the Ectocervical Epithelium.

Background: Genital mucosa is the main portal of entry for various incoming pathogens, including human immunodeficiency virus (HIV), hence it is an important site for host immune defenses. Tissue-resident memory T (TRM) cells defend tissue barriers against infections and are characterized by expression of CD103 and CD69. In this study, we describe the composition of CD8+ TRM cells in the ectocervix of healthy and HIV-infected women. Methods: Study samples were collected from healthy Swedish and Kenyan HIV-infected and uninfected women. Customized computerized image-based in situ analysis was developed to assess the ectocervical biopsies. Genital mucosa and blood samples were assessed by flow cytometry. Results: Although the ectocervical epithelium of healthy women was populated with bona fide CD8+ TRM cells (CD103+CD69+), women infected with HIV displayed a high frequency of CD103-CD8+ cells residing close to their epithelial basal membrane. Accumulation of CD103-CD8+ cells was associated with chemokine expression in the ectocervix and HIV viral load. CD103+CD8+ and CD103-CD8+ T cells expressed cytotoxic effector molecules in the ectocervical epithelium of healthy and HIV-infected women. In addition, women infected with HIV had decreased frequencies of circulating CD103+CD8+ T cells. Conclusions: Our data provide insight into the distribution of CD8+ TRM cells in human genital mucosa, a critically important location for immune defense against pathogens, including HIV.

Perturbed CD8+ T cell TIGIT/CD226/PVR axis despite early initiation of antiretroviral treatment in HIV infected individuals.

HIV-specific CD8+ T cells demonstrate an exhausted phenotype associated with increased expression of inhibitory receptors, decreased functional capacity, and a skewed transcriptional profile, which are only partially restored by antiretroviral treatment (ART). Expression levels of the inhibitory receptor, T cell immunoglobulin and ITIM domain (TIGIT), the co-stimulatory receptor CD226 and their ligand PVR are altered in viral infections and cancer. However, the extent to which the TIGIT/CD226/PVR-axis is affected by HIV-infection has not been characterized. Here, we report that TIGIT expression increased over time despite early initiation of ART. HIV-specific CD8+ T cells were almost exclusively TIGIT+, had an inverse expression of the transcription factors T-bet and Eomes and co-expressed PD-1, CD160 and 2B4. HIV-specific TIGIThi cells were negatively correlated with polyfunctionality and displayed a diminished expression of CD226. Furthermore, expression of PVR was increased on CD4+ T cells, especially T follicular helper (Tfh) cells, in HIV-infected lymph nodes. These results depict a skewing of the TIGIT/CD226 axis from CD226 co-stimulation towards TIGIT-mediated inhibition of CD8+ T cells, despite early ART. These findings highlight the importance of the TIGIT/CD226/PVR axis as an immune checkpoint barrier that could hinder future "cure" strategies requiring potent HIV-specific CD8+ T cells.

Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency.

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)-related diseases. Three patients presented with EBV-associated Hodgkin's lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro-generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70-CD27 interactions therefore play a nonredundant role in T and B cell-mediated immunity, especially for protection against EBV and humoral immunity.

CD4+ T cells with an activated and exhausted phenotype distinguish immunodeficiency during aviremic HIV-2 infection.

OBJECTIVE: HIV type 2 (HIV-2) represents an attenuated form of HIV, in which many infected individuals remain 'aviremic' without antiretroviral therapy. However, aviremic HIV-2 disease progression exists, and in the current study, we therefore aimed to examine if specific pathological characteristics of CD4 T cells are linked to such outcome. DESIGN: HIV-seronegative (n = 25), HIV type 1 (HIV-1) (n = 33), HIV-2 (n = 39, of whom 26 were aviremic), and HIV-1/2 dually (HIV-D) (n = 13)-infected study participants were enrolled from an occupational cohort in Guinea-Bissau. METHODS: CD4 T-cell differentiation, activation, exhaustion, senescence, and transcription factors were assessed by polychromatic flow cytometry. Multidimensional clustering bioinformatic tools were used to identify CD4 T-cell subpopulations linked to infection type and disease stage. RESULTS: HIV-2-infected individuals had early and late-differentiated CD4 T-cell clusters with lower activation (CD38HLA-DR) and exhaustion programmed death-1 (PD-1) than HIV-1 and HIV-D-infected individuals. We also noted that aviremic HIV-2-infected individuals possessed fewer individuals. CD4 T cells with pathological signs compared to other HIV-infected groups. Still, compared to HIV-seronegative individuals, aviremic HIV-2-infected individuals had T-bet CD4 T cells that showed elevated immune activation/exhaustion, and particularly the frequencies of PD-1 cells were associated with a suboptimal percentage of CD4 T cells. CONCLUSION: Increased frequencies of CD4 T cells with an activated/exhausted phenotype correlate with exacerbated immunodeficiency in aviremic HIV-2-infected individuals. Thus, these findings encourage studies on the introduction of antiretroviral therapy also to individuals with aviremic HIV-2 infection.

Correction: Rapid Progressing Allele HLA-B35 Px Restricted Anti-HIV-1 CD8+ T Cells Recognize Vestigial CTL Epitopes.

[This corrects the article DOI: 10.1371/journal.pone.0010249.].

Elevated levels of invariant natural killer T-cell and natural killer cell activation correlate with disease progression in HIV-1 and HIV-2 infections.

OBJECTIVE: In this study, we aimed to investigate the frequency and activation of invariant natural killer T (iNKT) cells and natural killer (NK) cells among HIV-1, HIV-2, or dually HIV-1/HIV-2 (HIV-D)-infected individuals, in relation to markers of disease progression. DESIGN: Whole blood samples were collected from treatment-naive HIV-1 (n = 23), HIV-2 (n = 34), and HIV-D (n = 11) infected individuals, as well as HIV-seronegative controls (n = 25), belonging to an occupational cohort in Guinea-Bissau. METHODS: Frequencies and activation levels of iNKT and NK cell subsets were analysed using multicolour flow cytometry, and results were related to HIV-status, CD4 T-cell levels, viral load, and T-cell activation. RESULTS: HIV-1, HIV-D, and viremic HIV-2 individuals had lower numbers of CD4 iNKT cells in circulation compared with seronegative controls. Numbers of CD56 NK cells were also reduced in HIV-infected individuals as compared with control study participants. Notably, iNKT cell and NK cell activation levels, assessed by CD38 expression, were increased in HIV-1 and HIV-2 single, as well as dual, infections. HIV-2 viremia was associated with elevated activation levels in CD4 iNKT cells, CD56, and CD56 NK cells, as compared with aviremic HIV-2 infection. Additionally, disease markers such as CD4 T-cell percentages, viral load, and CD4 T-cell activation were associated with CD38 expression levels of both iNKT and NK cells, which activation levels also correlated with each other. CONCLUSION: Our data indicate that elevated levels of iNKT-cell and NK-cell activation are associated with viremia and disease progression markers in both HIV-1 and HIV-2 infections.

Multidimensional Clusters of CD4+ T Cell Dysfunction Are Primarily Associated with the CD4/CD8 Ratio in Chronic HIV Infection.

HIV infection provokes a myriad of pathological effects on the immune system where many markers of CD4+ T cell dysfunction have been identified. However, most studies to date have focused on single/double measurements of immune dysfunction, while the identification of pathological CD4+ T cell clusters that is highly associated to a specific biomarker for HIV disease remain less studied. Here, multi-parametric flow cytometry was used to investigate immune activation, exhaustion, and senescence of diverse maturation phenotypes of CD4+ T cells. The traditional method of manual data analysis was compared to a multidimensional clustering tool, FLOw Clustering with K (FLOCK) in two cohorts of 47 untreated HIV-infected individuals and 21 age and sex matched healthy controls. In order to reduce the subjectivity of FLOCK, we developed an "artificial reference", using 2% of all CD4+ gated T cells from each of the HIV-infected individuals. Principle component analyses demonstrated that using an artificial reference lead to a better separation of the HIV-infected individuals from the healthy controls as compared to using a single HIV-infected subject as a reference or analyzing data manually. Multiple correlation analyses between laboratory parameters and pathological CD4+ clusters revealed that the CD4/CD8 ratio was the preeminent surrogate marker of CD4+ T cells dysfunction using all three methods. Increased frequencies of an early-differentiated CD4+ T cell cluster with high CD38, HLA-DR and PD-1 expression were best correlated (Rho = -0.80, P value = 1.96×10-11) with HIV disease progression as measured by the CD4/CD8 ratio. The novel approach described here can be used to identify cell clusters that distinguish healthy from HIV infected subjects and is biologically relevant for HIV disease progression. These results further emphasize that a simple measurement of the CD4/CD8 ratio is a useful biomarker for assessment of combined CD4+ T cell dysfunction in chronic HIV disease.

Single-Cell Characterization of in vitro Migration and Interaction Dynamics of T Cells Expanded with IL-2 and IL-7.

T cells are pivotal in the immune defense against cancers and infectious agents. To mount an effector response against cancer cells, T cells need to migrate to the cancer-site, engage in contacts with cancer cells, and perform their effector functions. Adoptive T cell therapy is an effective strategy as treatment of complications such as relapse or opportunistic infections after hematopoietic stem cell transplantations. This requires a sufficient amount of cells that are able to expand and respond to tumor or viral antigens. The cytokines interleukin (IL)-2 and IL-7 drive T cell differentiation, proliferation, and survival and are commonly used to expand T cells ex vivo. Here, we have used microchip-based live-cell imaging to follow the migration of individual T cells, their interactions with allogeneic monocytes, cell division, and apoptosis for extended periods of time; something that cannot be achieved by commonly used methods. Our data indicate that cells grown in IL-7 + IL-2 had similar migration and contact dynamics as cells grown in IL-2 alone. However, the addition of IL-7 decreased cell death creating a more viable cell population, which should be beneficial when preparing cells for immunotherapy.

Baseline CD4+ T cell counts correlates with HIV-1 synonymous rate in HLA-B*5701 subjects with different risk of disease progression.

HLA-B*5701 is the host factor most strongly associated with slow HIV-1 disease progression, although risk of progression may vary among patients carrying this allele. The interplay between HIV-1 evolutionary rate variation and risk of progression to AIDS in HLA-B*5701 subjects was studied using longitudinal viral sequences from high-risk progressors (HRPs) and low-risk progressors (LRPs). Posterior distributions of HIV-1 genealogies assuming a Bayesian relaxed molecular clock were used to estimate the absolute rates of nonsynonymous and synonymous substitutions for different set of branches. Rates of viral evolution, as well as in vitro viral replication capacity assessed using a novel phenotypic assay, were correlated with various clinical parameters. HIV-1 synonymous substitution rates were significantly lower in LRPs than HRPs, especially for sets of internal branches. The viral population infecting LRPs was also characterized by a slower increase in synonymous divergence over time. This pattern did not correlate to differences in viral fitness, as measured by in vitro replication capacity, nor could be explained by differences among subjects in T cell activation or selection pressure. Interestingly, a significant inverse correlation was found between baseline CD4+ T cell counts and mean HIV-1 synonymous rate (which is proportional to the viral replication rate) along branches representing viral lineages successfully propagating through time up to the last sampled time point. The observed lower replication rate in HLA-B*5701 subjects with higher baseline CD4+ T cell counts provides a potential model to explain differences in risk of disease progression among individuals carrying this allele.

NetFCM: a semi-automated web-based method for flow cytometry data analysis.

Multi-parametric flow cytometry (FCM) represents an invaluable instrument to conduct single cell analysis and has significantly increased our understanding of the immune system. However, due to new techniques allowing us to measure an increased number of phenotypes within the immune system, FCM data analysis has become more complex and labor-intensive than previously. We have therefore developed a semi-automatic gating strategy (NetFCM) that uses clustering and principal component analysis (PCA) together with other statistical methods to mimic manual gating approaches. NetFCM is an online tool both for subset identification as well as for quantification of differences between samples. Additionally, NetFCM can classify and cluster samples based on multidimensional data. We tested the method using a data set of peripheral blood mononuclear cells collected from 23 HIV-infected individuals, which were stimulated with overlapping HIV Gag-p55 and CMV-pp65 peptides or medium alone (negative control). NetFCM clustered the virus-specific CD8+ T cells based on IFNγ and TNF responses into distinct compartments. Additionally, NetFCM was capable of identifying HIV- and CMV-specific responses corresponding to those obtained by manual gating strategies. These data demonstrate that NetFCM has the potential to identify relevant T cell populations by mimicking classical FCM data analysis and reduce the subjectivity and amount of time associated with such analysis. © 2014 International Society for Advancement of Cytometry.

T-bet and Eomes are differentially linked to the exhausted phenotype of CD8+ T cells in HIV infection.

CD8(+) T cell exhaustion represents a major hallmark of chronic HIV infection. Two key transcription factors governing CD8(+) T cell differentiation, T-bet and Eomesodermin (Eomes), have previously been shown in mice to differentially regulate T cell exhaustion in part through direct modulation of PD-1. Here, we examined the relationship between these transcription factors and the expression of several inhibitory receptors (PD-1, CD160, and 2B4), functional characteristics and memory differentiation of CD8(+) T cells in chronic and treated HIV infection. The expression of PD-1, CD160, and 2B4 on total CD8(+) T cells was elevated in chronically infected individuals and highly associated with a T-bet(dim)Eomes(hi) expressional profile. Interestingly, both resting and activated HIV-specific CD8(+) T cells in chronic infection were almost exclusively T-bet(dim)Eomes(hi) cells, while CMV-specific CD8(+) T cells displayed a balanced expression pattern of T-bet and Eomes. The T-bet(dim)Eomes(hi) virus-specific CD8(+) T cells did not show features of terminal differentiation, but rather a transitional memory phenotype with poor polyfunctional (effector) characteristics. The transitional and exhausted phenotype of HIV-specific CD8(+) T cells was longitudinally related to persistent Eomes expression after antiretroviral therapy (ART) initiation. Strikingly, these characteristics remained stable up to 10 years after ART initiation. This study supports the concept that poor human viral-specific CD8(+) T cell functionality is due to an inverse expression balance between T-bet and Eomes, which is not reversed despite long-term viral control through ART. These results aid to explain the inability of HIV-specific CD8(+) T cells to control the viral replication post-ART cessation.