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1. Microfluidic Assays for CD4 T Lymphocyte Counting: A Review

   https://doi.org/10.3390/bios15010033  

   Meng, Zhuolun; Raji, Hassan; Kokabi, Mahtab; Zou, Deng; Chan, James; Liu, Qihao; Zhang, Ruifeng; Javanmard, Mehdi (January 2025, Biosensors)

   CD4 T lymphocytes play a key role in initiating the adaptive immune response, releasing cytokines that mediate numerous signal transduction pathways across the immune system. Therefore, CD4 T cell counts are widely used as an indicator of overall immunological health. HIV, one of the leading causes of death in the developing world, specifically targets and gradually depletes CD4 cells, making CD4 counts a critical metric for monitoring disease progression. As a result, accurately counting CD4 cells represents a pressing challenge in global healthcare. Flow cytometry remains the gold standard for enumerating CD4 T cells; however, flow cytometers are expensive, difficult to transport, and require skilled medical staff to prepare samples, operate the equipment, and interpret results. This highlights the critical need for novel, rapid, cost-effective, and portable methods of CD4 enumeration that are suitable for deployment in resource-limited countries. This review will survey and analyze emerging research in CD4 counting, with a focus on microfluidic systems, which represent a promising area of investigation. 

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2. Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses

   https://doi.org/10.1038/s41592-021-01328-8  

   Albanese, Manuel; Ruhle, Adrian; Mittermaier, Jennifer; Mejías-Pérez, Ernesto; Gapp, Madeleine; Linder, Andreas; Schmacke, Niklas A.; Hofmann, Katharina; Hennrich, Alexandru A.; Levy, David N.; et al (December 2021, Nature Methods)

   Abstract CD4 + T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4 + T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4 + T cells using optimized cell cultivation and nucleofection conditions of Cas9–guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4 + T cells. 

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3. A click chemistry amplified nanopore assay for ultrasensitive quantification of HIV-1 p24 antigen in clinical samples

   https://doi.org/10.1038/s41467-022-34273-x  

   Wei, Xiaojun; Wang, Xiaoqin; Zhang, Zehui; Luo, Yuanyuan; Wang, Zixin; Xiong, Wen; Jain, Piyush K.; Monnier, John R.; Wang, Hui; Hu, Tony Y.; et al (November 2022, Nature Communications)

   Abstract Despite major advances in HIV testing, ultrasensitive detection of early infection remains challenging, especially for the viral capsid protein p24, which is an early virological biomarker of HIV-1 infection. Here, To improve p24 detection in patients missed by immunological tests that dominate the diagnostics market, we show a click chemistry amplified nanopore (CAN) assay for ultrasensitive quantitative detection. This strategy achieves a 20.8 fM (0.5 pg/ml) limit of detection for HIV-1 p24 antigen in human serum, demonstrating 20~100-fold higher analytical sensitivity than nanocluster-based immunoassays and clinically used enzyme-linked immunosorbent assay, respectively. Clinical validation of the CAN assay in a pilot cohort shows p24 quantification at ultra-low concentration range and correlation with CD4 count and viral load. We believe that this strategy can improve the utility of p24 antigen in detecting early infection and monitoring HIV progression and treatment efficacy, and also can be readily modified to detect other infectious diseases. 

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4. Extrafollicular CD4 T cell-derived IL-10 functions rapidly and transiently to support anti-Plasmodium humoral immunity

   https://doi.org/10.1371/journal.ppat.1009288  

   Surette, Fionna A.; Guthmiller, Jenna J.; Li, Lei; Sturtz, Alexandria J.; Vijay, Rahul; Pope, Rosemary L.; McClellan, Brandon L.; Pack, Angela D.; Zander, Ryan A.; Shao, Peng; et al (February 2021, PLOS Pathogens)

   Higgins, Matthew K. (Ed.)

   Immunity against malaria depends on germinal center (GC)-derived antibody responses that are orchestrated by T follicular helper (TFH) cells. Emerging data show that the regulatory cytokine IL-10 plays an essential role in promoting GC B cell responses during both experimental malaria and virus infections. Here we investigated the cellular source and temporal role of IL-10, and whether IL-10 additionally signals to CD4 T-cells to support anti- Plasmodium humoral immunity. Distinct from reports of virus infection, we found that IL-10 was expressed by conventional, Foxp3-negative effector CD4 T cells and functioned in a B cell-intrinsic manner only during the first 96 hours of Plasmodium infection to support humoral immunity. The critical functions of IL-10 manifested only before the orchestration of GC responses and were primarily localized outside of B cell follicles. Mechanistically, our studies showed that the rapid and transient provision of IL-10 promoted B cell expression of anti-apoptotic factors, MHC class II, CD83, and cell-cell adhesion proteins that are essential for B cell survival and interaction with CD4 T cells. Together, our data reveal temporal features and mechanisms by which IL-10 critically supports humoral immunity during blood-stage Plasmodium infection, information that may be useful for developing new strategies designed to lessen the burden of malaria. 

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5. Intact proviral DNA assay analysis of large cohorts of people with HIV provides a benchmark for the frequency and composition of persistent proviral DNA

   https://doi.org/10.1073/pnas.2006816117  

   Simonetti, Francesco R.; White, Jennifer A.; Tumiotto, Camille; Ritter, Kristen D.; Cai, Mian; Gandhi, Rajesh T.; Deeks, Steven G.; Howell, Bonnie J.; Montaner, Luis J.; Blankson, Joel N.; et al (August 2020, Proceedings of the National Academy of Sciences)

   A scalable approach for quantifying intact HIV-1 proviruses is critical for basic research and clinical trials directed at HIV-1 cure. The intact proviral DNA assay (IPDA) is a novel approach to characterizing the HIV-1 reservoir, focusing on the genetic integrity of individual proviruses independent of transcriptional status. It uses multiplex digital droplet PCR to distinguish and separately quantify intact proviruses, defined by a lack of overt fatal defects such as large deletions and APOBEC3G-mediated hypermutation, from the majority of proviruses that have such defects. This distinction is important because only intact proviruses cause viral rebound on ART interruption. To evaluate IPDA performance and provide benchmark data to support its implementation, we analyzed peripheral blood samples from 400 HIV-1 + adults on ART from several diverse cohorts, representing a robust sample of treated HIV-1 infection in the United States. We provide direct quantitative evidence that defective proviruses greatly outnumber intact proviruses (by >12.5 fold). However, intact proviruses are present at substantially higher frequencies (median, 54/10 6 CD4 + T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (∼1/10 6 CD4 + T cells). IPDA amplicon signal issues resulting from sequence polymorphisms were observed in only 6.3% of individuals and were readily apparent and easily distinguished from low proviral frequency, an advantage of the IPDA over standard PCR assays which generate false-negative results in such situations. The large IPDA dataset provided here gives the clearest quantitative picture to date of HIV-1 proviral persistence on ART. 

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