A VRC13-like bNAb response is associated with complex escape pathways in HIV-1 envelope.

The rational design of HIV-1 immunogens to trigger the development of broadly neutralizing antibodies (bNAbs) requires understanding the viral evolutionary pathways influencing this process. An acute HIV-1-infected individual exhibiting >50% plasma neutralization breadth developed neutralizing antibody specificities against the CD4-binding site (CD4bs) and V1V2 regions of Env gp120. Comparison of pseudoviruses derived from early and late autologous env sequences demonstrated the development of >2 log resistance to VRC13 but not to other CD4bs-specific bNAbs. Mapping studies indicated that the V3 and CD4-binding loops of Env gp120 contributed significantly to developing resistance to the autologous neutralizing response and that the CD4-binding loop (CD4BL) specifically was responsible for the developing resistance to VRC13. Tracking viral evolution during the development of this cross-neutralizing CD4bs response identified amino acid substitutions arising at only 4 of 11 known VRC13 contact sites (K282, T283, K421, and V471). However, each of these mutations was external to the V3 and CD4BL regions conferring resistance to VRC13 and was transient in nature. Rather, complete resistance to VRC13 was achieved through the cooperative expression of a cluster of single amino acid changes within and immediately adjacent to the CD4BL, including a T359I substitution, exchange of a potential N-linked glycosylation (PNLG) site to residue S362 from N363, and a P369L substitution. Collectively, our data characterize complex HIV-1 env evolution in an individual developing resistance to a VRC13-like neutralizing antibody response and identify novel VRC13-associated escape mutations that may be important to inducing VRC13-like bNAbs for lineage-based immunogens.IMPORTANCEThe pursuit of eliciting broadly neutralizing antibodies (bNAbs) through vaccination and their use as therapeutics remains a significant focus in the effort to eradicate HIV-1. Key to our understanding of this approach is a more extensive understanding of bNAb contact sites and susceptible escape mutations in HIV-1 envelope (env). We identified a broad neutralizer exhibiting VRC13-like responses, a non-germline restricted class of CD4-binding site antibody distinct from the well-studied VRC01-class. Through longitudinal envelope sequencing and Env-pseudotyped neutralization assays, we characterized a complex escape pathway requiring the cooperative evolution of four amino acid changes to confer complete resistance to VRC13. This suggests that VRC13-class bNAbs may be refractory to rapid escape and attractive for therapeutic applications. Furthermore, the identification of longitudinal viral changes concomitant with the development of neutralization breadth may help identify the viral intermediates needed for the maturation of VRC13-like responses and the design of lineage-based immunogens.

Inactivation of cell-free HIV-1 by designing potent peptides based on mutations in the CD4 binding site.

Human immunodeficiency virus type 1 (HIV-1) is a major global health problem, with over 38 million people infected worldwide. Current anti-HIV-1 drugs are limited in their ability to prevent the virus from replicating inside host cells, making them less effective as preventive measures. In contrast, viral inhibitors that inactivate the virus before it can bind to a host cell have great potential as drugs. In this study, we aimed to design mutant peptides that could block the interaction between gp120 and the CD4 receptor on host cells, thus preventing HIV-1 infection. We designed a 20-amino-acid peptide that mimicked the amino acids of the CD4 binding site and docked it to gp120. Molecular dynamics simulations were performed to calculate the energy of MMPBSA (Poisson-Boltzmann Surface Area) for each residue of the peptide, and unfavorable energy residues were identified as potential mutation points. Using MAESTRO (Multi AgEnt STability pRedictiOn), we measured ΔΔG (change in the change in Gibbs free energy) for mutations and generated a library of 240 mutated peptides using OSPREY software. The peptides were then screened for allergenicity and binding affinity. Finally, molecular dynamics simulations (via GROMACS 2020.2) and control docking (via HADDOCK 2.4) were used to evaluate the ability of four selected peptides to inhibit HIV-1 infection. Three peptides, P3 (AHRQIRQWFLTRGPNRSLWQ), P4 (VHRQIRQWFLTRGPNRSLWQ), and P9 (AHRQIRQMFLTRGPNRSLWQ), showed practical and potential as HIV inhibitors, based on their binding affinity and ability to inhibit infection. These peptides have the ability to inactivate the virus before it can bind to a host cell, thus representing a promising approach to HIV-1 prevention. Our findings suggest that mutant peptides designed to block the interaction between gp120 and the CD4 receptor have potential as HIV-1 inhibitors. These peptides could be used as preventive measures against HIV-1 transmission, and further research is needed to evaluate their safety and efficacy in clinical settings.

Potent latency reversal by Tat RNA-containing nanoparticle enables multi-omic analysis of the HIV-1 reservoir.

The development of latency reversing agents that potently reactivate HIV without inducing global T cell activation would benefit the field of HIV reservoir research and could pave the way to a functional cure. Here, we explore the reactivation capacity of a lipid nanoparticle containing Tat mRNA (Tat-LNP) in CD4 T cells from people living with HIV undergoing antiretroviral therapy (ART). When combined with panobinostat, Tat-LNP induces latency reversal in a significantly higher proportion of latently infected cells compared to PMA/ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the transcriptome of CD4 T cells, enabling the characterization of latently infected cells in their near-native state. Upon latency reversal, we identify transcriptomic differences between infected cells carrying an inducible provirus and non-infected cells (e.g. LINC02964, GZMA, CCL5). We confirm the transcriptomic differences at the protein level and provide evidence that the long non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore, p24+ cells exhibit heightened PI3K/Akt signaling, along with downregulation of protein translation, suggesting that HIV-infected cells display distinct signatures facilitating their long-term persistence. Tat-LNP represents a valuable research tool for in vitro reservoir studies as it greatly facilitates the in-depth characterization of HIV reservoir cells' transcriptome and proteome profiles.

Lack of Association between Common LAG3/CD4 Variants and Risk of Migraine.

Several papers have been published suggesting a probable role of inflammatory factors in the etiopathogenesis of migraine. In this study, we investigated the possible association between common variants in the LAG3/CD4 genes (both genes, which are closely related, encode proteins involved in inflammatory and autoimmune responses) in the risk of migraine in a cohort of Caucasian Spanish participants. For this purpose, the frequencies of CD4 rs1922452, CD4 rs951818, and LAG3 rs870849 genotypes and allelic variants, using a specific TaqMan-based qPCR assay, were assessed in 290 patients diagnosed with migraine and in 300 healthy controls. The relationship of these variables with several clinical features of migraine was also analyzed. The frequencies of the analyzed LAG3/CD4 genotypes did not differ significantly between the two study groups and were not related to the sex, age at onset of migraine, family history of migraine, presence or absence of aura, or the triggering effect of ethanol on migraine episodes. These results suggest a lack of association between common variants in the LAG3/CD4 genes and the risk of developing migraine in the Caucasian Spanish population.

LAG3/CD4 Genes Variants and the Risk for Restless Legs Syndrome.

According to several studies, inflammatory factors could be related to the pathogenesis of idiopathic restless legs syndrome (RLS). In addition, RLS and Parkinson's disease (PD) have shown a possible relationship, and recent studies have shown an association between CD4 rs1922452 and CD4 rs951818 single nucleotide variants (SNVs) and the risk for PD. For these reasons, we investigated the possible association between common variants in the LAG3/CD4 genes (which encoded proteins involved in inflammatory and autoimmune responses) and the risk for RLS in a Caucasian Spanish population. We assessed the frequencies of CD4 rs1922452, CD4 rs951818, and LAG3 rs870849 genotypes and allelic variants in 285 patients with idiopathic RLS and 350 healthy controls using a specific TaqMan-based qPCR assay. We also analyzed the possible influence of the genotypes' frequencies on several variables, including age at onset of RLS, gender, family history of RLS, and response to drugs commonly used in the treatment of RLS. We found a lack of association between the frequencies of genotypes and allelic variants of the 3 SNVs studied and the risk of RLS, and a weak though significant association between the CD4 rs1922452 GG genotype and an older age at onset of RLS. With the exception of this association, our findings suggest that common SNVs in the CD4/LAG3 genes are not associated with the risk of developing idiopathic RLS in Caucasian Spanish people.

Chromatin organizer SATB1 controls the cell identity of CD4+ CD8+ double-positive thymocytes by regulating the activity of super-enhancers.

CD4+ and CD8+ double-positive (DP) thymocytes play a crucial role in T cell development in the thymus. DP cells rearrange the T cell receptor gene Tcra to generate T cell receptors with TCRß. DP cells differentiate into CD4 or CD8 single-positive (SP) thymocytes, regulatory T cells, or invariant nature kill T cells (iNKT) in response to TCR signaling. Chromatin organizer SATB1 is highly expressed in DP cells and is essential in regulating Tcra rearrangement and differentiation of DP cells. Here we explored the mechanism of SATB1 orchestrating gene expression in DP cells. Single-cell RNA sequencing shows that Satb1 deletion changes the cell identity of DP thymocytes and down-regulates genes specifically and highly expressed in DP cells. Super-enhancers regulate the expressions of DP-specific genes, and our Hi-C data show that SATB1 deficiency in thymocytes reduces super-enhancer activity by specifically decreasing interactions among super-enhancers and between super-enhancers and promoters. Our results reveal that SATB1 plays a critical role in thymocyte development to promote the establishment of DP cell identity by globally regulating super-enhancers of DP cells at the chromatin architectural level.

CD4 bovine gene: Differential polymorphisms among cattle breeds and a new tool for rapid identification.

Two mutations in the CD4 bovine gene (G>T/Q306H; A>C/K310N) were identified as causative for altered staining with anti-CD4 mAb #CC8. We developed a HRM qPCR for genotyping these mutations and compare with immunophenotyping in different cattle breeds. The assay distinguished five genotypes, B (homozygous, G/A) and C (heterozygous, G/A and T/C), found in taurine, A (homozygous, G/C) and D (heterozygous, T/C and G/C), found in zebu. The E genotype (homozygous, T/C) was not observed in tested animals. As expected, B and C presented high/very high and intermediate CD4 staining, respectively. The lack/low CD4 staining was mainly related to the A, while the intermediate staining was mainly related to D genotype. The developed HRM qPCR assay accurately identified the altered phenotypes associated with CC8 staining in taurine. However, the assay cannot be applicable in zebu or hybrid breeds, probably due to additional mutations in the CD4 gene from zebu descendant animals.

Enhancing and inhibitory motifs regulate CD4 activity.

CD4+ T cells use T cell receptor (TCR)-CD3 complexes, and CD4, to respond to peptide antigens within MHCII molecules (pMHCII). We report here that, through ~435 million years of evolution in jawed vertebrates, purifying selection has shaped motifs in the extracellular, transmembrane, and intracellular domains of eutherian CD4 that enhance pMHCII responses, and covary with residues in an intracellular motif that inhibits responses. Importantly, while CD4 interactions with the Src kinase, Lck, are viewed as key to pMHCII responses, our data indicate that CD4-Lck interactions derive their importance from the counterbalancing activity of the inhibitory motif, as well as motifs that direct CD4-Lck pairs to specific membrane compartments. These results have implications for the evolution and function of complex transmembrane receptors and for biomimetic engineering.

A novel CD4 knockout mouse strain with a spontaneous frameshift mutation in the CD4 locus.

T cells express co-receptors CD4 and CD8, which are involved in the recognition of antigen presented to T cell receptors. The expression of CD4 in thymic hematopoietic cells is crucial for the thymic development and selection of T cells. In this study, we identified a novel CD4 mutant allele that emerged spontaneously in our mouse colony. The frameshift mutation led to a truncated CD4 protein which failed to reach the plasma membrane resulting in impaired development of CD4+ helper T cells. The CRISPR mediated correction of mutant allele restored the membrane CD4 expression. Further, using an adoptive transfer of T cells, we show that this model is an ideal recipient mouse for the study of CD4+ T cells.

SRSF1 Deficiency Impairs the Late Thymocyte Maturation and the CD8 Single-Positive Lineage Fate Decision.

The underlying mechanisms of thymocyte development and lineage determination remain incompletely understood, and the emerging evidences demonstrated that RNA binding proteins (RBPs) are deeply involved in governing T cell fate in thymus. Serine/arginine-rich splicing factor 1 (SRSF1), as a classical splicing factor, is a pivotal RBP for gene expression in various biological processes. Our recent study demonstrated that SRSF1 plays essential roles in the development of late thymocytes by modulating the T cell regulatory gene networks post-transcriptionally, which are critical in response to type I interferon signaling for supporting thymocyte maturation. Here, we report SRSF1 also contributes to the determination of the CD8+ T cell fate. By specific ablation of SRSF1 in CD4+CD8+ double positive (DP) thymocytes, we found that SRSF1 deficiency impaired the maturation of late thymocytes and diminished the output of both CD4+ and CD8+ single positive T cells. Interestingly, the ratio of mature CD4+ to CD8+ cells was notably altered and more severe defects were exhibited in CD8+ lineage than those in CD4+ lineage, reflecting the specific function of SRSF1 in CD8+ T cell fate decision. Mechanistically, SRSF1-deficient cells downregulate their expression of Runx3, which is a crucial transcriptional regulator in sustaining CD8+ single positive (SP) thymocyte development and lineage choice. Moreover, forced expression of Runx3 partially rectified the defects in SRSF1-deficient CD8+ thymocyte maturation. Thus, our data uncovered the previous unknown role of SRSF1 in establishment of CD8+ cell identity.

Deciphering gp120 sequence variation and structural dynamics in HIV neutralization phenotype by molecular dynamics simulations and graph machine learning.

Human immunodeficiency virus (HIV) exploits the sequence variation and structural dynamics of the envelope glycoprotein gp120 to evade the immune attack of neutralization antibodies, contributing to various HIV neutralization phenotypes. Although the HIV neutralization phenotype has been experimentally characterized, the roles of rapid sequence variability and significant structural dynamics of gp120 are not well understood. Here, 45 prefusion gp120 from different HIV strains belong to three tiers of sensitive, moderate, and resistant neutralization phenotype are structurally modeled by homology modeling and then investigated by molecular dynamics (MD) simulations and graph machine learning (ML). Our results show that the structural deviations, population distribution, and conformational flexibility of gp120 are related to the HIV neutralization phenotype. Per-residue dynamics indicate the local regions especially in the second structural elements with high-flexibility, may be responsible for the HIV neutralization phenotype. Moreover, a graph ML model with the attention mechanism was trained to explore inherent representation related to the classification of the HIV neutralization phenotype, further distinguishing the strong related gp120 sequence variation together with structural dynamics in the HIV neutralization phenotype. Our study not only deciphers gp120 sequence variation and structural dynamics in the HIV neutralization phenotype but also explores complex relationships between the sequence, structure, and dynamics of protein by combining MD simulations and ML.

Immunohistochemical analysis of CD68, CD4, CD8 and CD20 expression in cervical dysplasia and its relationship with HR-HPV infection.

OBJECTIVE: The aim of the study was to examine the composition of the inflammatory infiltrates in cervical premalignant lesions and contribute to a better understanding of immune response to HR-HPV infection and dysplasia. PATIENTS AND METHODS: Semi-quantitative analysis of CD68, CD4, CD8 and CD20 immunohistochemical expression in a series of 41 cervical biopsies without dysplasia, 24 cases of LSIL and 35 HSIL cases was performed. In each subject, genotyping for 12 HR-HPV types was done prior to the biopsy. RESULTS: Observing the total sample, no correlation between CD68, CD4, CD8 and CD20 expression levels and HR-HPV infection was found, regardless of the presence of mono- or co-infection (p>0.05). A statistically significant correlation between dysplastic changes and CD68 expression, as well as between dysplastic changes and CD4 expression, was observed (p=0.003 and p=0.016, respectively). For CD68 expression, there was a positive correlation with both LSIL and HSIL, and concerning CD4 expression, there was a positive correlation primarily with LSIL. The finding of mild CD68 expression shows a 10.5 times greater chance of the sample being classified as LSIL, while the finding of a strong CD68 expression shows a 12 times greater chance of the sample being classified as HSIL, in comparison to cases with no expression. When the samples were stratified in relation to the lesion grade, a correlation between HR-HPV infection and CD68/CD4 expression again was not proved (p>0.05). No correlation between CD8 and CD20 expression with dysplasia was found (p>0.05). CONCLUSIONS: We consider a higher prevalence of macrophages and CD4 lymphocytes in dysplastic lesions to be a response to dysplasia rather than HR-HPV infection itself. The increase of the expression levels of macrophages with the degree of the lesion speaks in favour of their potential role in the progression of the neoplastic process.

Understanding Viral and Immune Interplay During Vertical Transmission of HIV: Implications for Cure.

Despite the significant progress that has been made to eliminate vertical HIV infection, more than 150,000 children were infected with HIV in 2019, emphasizing the continued need for sustainable HIV treatment strategies and ideally a cure for children. Mother-to-child-transmission (MTCT) remains the most important route of pediatric HIV acquisition and, in absence of prevention measures, transmission rates range from 15% to 45% via three distinct routes: in utero, intrapartum, and in the postnatal period through breastfeeding. The exact mechanisms and biological basis of these different routes of transmission are not yet fully understood. Some infants escape infection despite significant virus exposure, while others do not, suggesting possible maternal or fetal immune protective factors including the presence of HIV-specific antibodies. Here we summarize the unique aspects of HIV MTCT including the immunopathogenesis of the different routes of transmission, and how transmission in the antenatal or postnatal periods may affect early life immune responses and HIV persistence. A more refined understanding of the complex interaction between viral, maternal, and fetal/infant factors may enhance the pursuit of strategies to achieve an HIV cure for pediatric populations.

Identifying the Main Functional Pathways Associated with Cognitive Resilience to Alzheimer's Disease.

Understanding the mechanisms involved in cognitive resilience in Alzheimer's disease (AD) represents a promising strategy to identify novel treatments for dementia in AD. Previous findings from our group revealed that the study of aged-Tg2576 cognitive resilient individuals is a suitable tool for this purpose. In the present study, we performed a transcriptomic analysis using the prefrontal cortex of demented and resilient Tg2576 transgenic AD mice. We have been able to hypothesize that pathways involved in inflammation, amyloid degradation, memory function, and neurotransmission may be playing a role on cognitive resilience in AD. Intriguingly, the results obtained in this study are suggestive of a reduction of the influx of peripheral immune cells into the brain on cognitive resilient subjects. Indeed, CD4 mRNA expression is significantly reduced on Tg2576 mice with cognitive resilience. For further validation of this result, we analyzed CD4 expression in human AD samples, including temporal cortex and peripheral blood mononuclear cells (PBMC). Interestingly, we have found a negative correlation between CD4 mRNA levels in the periphery and the score in the Mini-Mental State Exam of AD patients. These findings highlight the importance of understanding the role of the immune system on the development of neurodegenerative diseases and points out to the infiltration of CD4+ cells in the brain as a key player of cognitive dysfunction in AD.

Across Functional Boundaries: Making Nonneutralizing Antibodies To Neutralize HIV-1 and Mediate Fc-Mediated Effector Killing of Infected Cells.

In HIV-1 infection, many antibodies (Abs) are elicited to Envelope (Env) epitopes that are conformationally masked in the native trimer and are only available for antibody recognition after the trimer binds host cell CD4. Among these are epitopes within the Co-Receptor Binding Site (CoRBS) and the constant region 1 and 2 (C1-C2 or cluster A region). In particular, C1-C2 epitopes map to the gp120 face interacting with gp41 in the native, "closed" Env trimer present on HIV-1 virions or expressed on HIV-1-infected cells. Antibodies targeting this region are therefore nonneutralizing and their potential as mediators of antibody-dependent cellular cytotoxicity (ADCC) of HIV-1-infected cells diminished by a lack of available binding targets. Here, we present the design of Ab-CD4 chimeric proteins that consist of the Ab-IgG1 of a CoRBS or cluster A specificity to the extracellular domains 1 and 2 of human CD4. Our Ab-CD4 hybrids induce potent ADCC against infected primary CD4+ T cells and neutralize tier 1 and 2 HIV-1 viruses. Furthermore, competition binding experiments reveal that the observed biological activities rely on both the antibody and CD4 moieties, confirming their cooperativity in triggering conformational rearrangements of Env. Our data indicate the utility of these Ab-CD4 hybrids as antibody therapeutics that are effective in eliminating HIV-1 through the combined mechanisms of neutralization and ADCC. This is also the first report of single-chain-Ab-based molecules capable of opening "closed" Env trimers on HIV-1 particles/infected cells to expose the cluster A region and activate ADCC and neutralization against these nonneutralizing targets. IMPORTANCE Highly conserved epitopes within the coreceptor binding site (CoRBS) and constant region 1 and 2 (C1-C2 or cluster A) are only available for antibody recognition after the HIV-1 Env trimer binds host cell CD4; therefore, they are not accessible on virions and infected cells, where the expression of CD4 is downregulated. Here, we have developed new antibody fusion molecules in which domains 1 and 2 of soluble human CD4 are linked with monoclonal antibodies of either the CoRBS or cluster A specificity. We optimized the conjugation sites and linker lengths to allow each of these novel bispecific fusion molecules to recognize native "closed" Env trimers and induce the structural rearrangements required for exposure of the epitopes for antibody binding. Our in vitro functional testing shows that our Ab-CD4 molecules can efficiently target and eliminate HIV-1-infected cells through antibody-dependent cellular cytotoxicity and inactivate HIV-1 virus through neutralization.

Blastic Plasmacytoid Dendritic Cell Neoplasm: Progress in Cell Origin, Molecular Biology, Diagnostic Criteria and Therapeutic Approaches.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological malignancy characterized by recurrent skin nodules, an aggressive clinical course with rapid involvement of hematological organs, and a poor prognosis with poor overall survival. BPDCN is derived from plasmacytoid dendritic cells (pDCs) and its pathogenesis is unclear. The tumor cells show aberrant expression of CD4, CD56, interleukin-3 receptor alpha chain (CD123), blood dendritic cell antigen 2 (BDCA 2/CD303), blood dendritic cell antigen 4 (BDCA4) and transcription factor (E protein) E2-2 (TCF4). The best treatment drugs are based on experience by adopting those used for either leukemia or lymphoma. Relapse with drug resistance generally occurs quickly. Stem cell transplantation after the first complete remission is recommended and tagraxofusp is the first targeted therapy. In this review, we summarize the differentiation of BPDCN from its cell origin, its connection with normal pDCs, clinical characteristics, genetic mutations and advances in treatment of BPDCN. This review provides insights into the mechanisms of and new therapeutic approaches for BPDCN.

Increase of CD4+CD25highFoxP3+ cells impairs in vitro human microbicidal activity against Mycobacterium tuberculosis during latent and acute pulmonary tuberculosis.

BACKGROUND: Regulatory T cells (Tregs) play a critical role during Mycobacterium tuberculosis (Mtb) infection, modulating host responses while neutralizing excessive inflammation. However, their impact on regulating host protective immunity is not completely understood. Here, we demonstrate that Treg cells abrogate the in vitro microbicidal activity against Mtb. METHODS: We evaluated the in vitro microbicidal activity of peripheral blood mononuclear cells (PBMCs) from patients with active tuberculosis (TB), individuals with latent tuberculosis infection (LTBI, TST+/IGRA+) and healthy control (HC, TST-/IGRA-) volunteers. PBMCs, depleted or not of CD4+CD25+ T-cells, were analyzed to determine frequency and influence on microbicidal activity during in vitro Mtb infection with four clinical isolates (S1, S5, R3, and R6) and one reference strain (H37Rv). RESULTS: The frequency of CD4+CD25highFoxP3+ cells were significantly higher in Mtb infected whole blood cultures from both TB patients and LTBI individuals when compared to HC. Data from CD4+CD25+ T-cells depletion demonstrate that increase of CD4+CD25highFoxP3+ is associated with an impairment of Th-1 responses and a diminished in vitro microbicidal activity of LTBI and TB groups. CONCLUSIONS: Tregs restrict host anti-mycobacterial immunity during active disease and latent infection and thereby may contribute to both disease progression and pathogen persistence.

Lack of intrafollicular memory CD4 + T cells is predictive of early clinical failure in newly diagnosed follicular lymphoma.

Despite a characteristic indolent course, a substantial subset of follicular lymphoma (FL) patients has an early relapse with a poor outcome. Cells in the microenvironment may be a key contributor to treatment failure. We used a discovery and validation study design to identify microenvironmental determinants of early failure and then integrated these results into the FLIPI. In total, 496 newly diagnosed FL grade 1-3 A patients who were prospectively enrolled into the MER cohort from 2002 to 2012 were evaluated. Tissue microarrays were stained for CD4, CD8, FOXP3, CD32b, CD14, CD68, CD70, SIRP-α, TIM3, PD-1, and PD-L1. Early failure was defined as failing to achieve event-free survival at 24 months (EFS24) in immunochemotherapy-treated patients and EFS12 in all others. CyTOF and CODEX analysis were performed to characterize intratumoral immunophenotypes. Lack of intrafollicular CD4 expression was the only predictor of early failure that replicated with a pooled OR 2.37 (95%CI 1.48-3.79). We next developed a bio-clinical risk model (BioFLIPI), where lack of CD4 intrafollicular expression moved patients up one FLIPI risk group, adding a new fourth high-risk group. Compared with BioFLIPI score of 1, patients with a score of 2 (OR 2.17; 95% CI 1.08-4.69), 3 (OR 3.53; 95% CI 1.78-7.54), and 4 (OR 8.92; 95% CI 4.00-21.1) had increasing risk of early failure. The favorable intrafollicular CD4 T cells were identified as activated central memory T cells, whose prognostic value was independent from genetic features. In conclusion, lack of intrafollicular CD4 expression predicts early failure in FL and combined with FLIPI improves identification of high-risk patients; however, independent validation is warranted.

NKG2D Enhances Double-Negative T Cell Regulation of B Cells.

Numerous studies reported a small subpopulation of TCRαß+CD4-CD8- (double-negative) T cells that exert regulatory functions in the peripheral lymphocyte population. However, the origin of these double-negative T (DNT) cells is controversial. Some researchers reported that DNT cells originated from the thymus, and others argued that these cells are derived from peripheral immune induction. We report a possible mechanism for the induction of nonregulatory CD4+ T cells to become regulatory double-negative T (iDNT) cells in vitro. We found that immature bone marrow dendritic cells (CD86+MHC-II- DCs), rather than mature DCs (CD86+MHC-II+), induced high levels of iDNT cells. The addition of an anti-MHC-II antibody to the CD86+MHC-II+ DC group significantly increased induction. These iDNT cells promoted B cell apoptosis and inhibited B cell proliferation and plasma cell formation. A subgroup of iDNT cells expressed NKG2D. Compared to NKG2D- iDNT cells, NKG2D+ iDNT cells released more granzyme B to enhance B cell regulation. This enhancement may function via NKG2D ligands expressed on B cells following lipopolysaccharide stimulation. These results demonstrate that MHC-II impedes induction, and iDNT cells may be MHC independent. NKG2D expression on iDNT cells enhanced the regulatory function of these cells. Our findings elucidate one possible mechanism of the induction of peripheral immune tolerance and provide a potential treatment for chronic allograft rejection in the future.

Mucosal and Systemic Immune Responses to Salmon Gill Poxvirus Infection in Atlantic Salmon Are Modulated Upon Hydrocortisone Injection.

Salmon Gill Poxvirus Disease (SGPVD) has emerged as a cause of acute mortality in Atlantic salmon (Salmo salar L.) presmolts in Norwegian aquaculture. The clinical phase of the disease is associated with apoptotic cell death in the gill epithelium causing acute respiratory distress, followed by proliferative changes in the regenerating gill in the period after the disease outbreak. In an experimental SGPV challenge trial published in 2020, acute disease was only seen in fish injected with hydrocortisone 24 h prior to infection. SGPV-mediated mortality in the hydrocortisone-injected group was associated with more extensive gill pathology and higher SGPV levels compared to the group infected with SGPV only. In this study based on the same trial, SGPV gene expression and the innate and adaptive antiviral immune response was monitored in gills and spleen in the presence and absence of hydrocortisone. Whereas most SGPV genes were induced from day 3 along with the interferon-regulated innate immune response in gills, the putative SGPV virulence genes of the B22R family were expressed already one day after SGPV exposure, indicating a potential role as early markers of SGPV infection. In gills of the hydrocortisone-injected fish infected with SGPV, MX expression was delayed until day 10, and then expression skyrocketed along with the viral peak, gill pathology and mortality occurring from day 14. A similar expression pattern was observed for Interferon gamma (IFNγ) and granzyme A (GzmA) in the gills, indicating a role of acute cytotoxic cell activity in SGPVD. Duplex in situ hybridization demonstrated effects of hydrocortisone on the number and localization of GzmA-containing cells, and colocalization with SGPV infected cells in the gill. SGPV was generally not detected in spleen, and gill infection did not induce any corresponding systemic immune activity in the absence of stress hormone injection. However, in fish injected with hydrocortisone, IFNγ and GzmA gene expression was induced in spleen in the days prior to acute mortality. These data indicate that suppressed mucosal immune response in the gills and the late triggered systemic immune response in the spleen following hormonal stress induction may be the key to the onset of clinical SGPVD.