Microfluidic 3D Cytotoxic Assay.

Microfluidic-based cytotoxic assays provide high physiological relevance with the potential to replace conventional animal experiments and two-dimensional (2D) assays. Here, a 3D method utilizing a microfluidic platform for analysis of lymphocyte cytotoxicity is introduced in detail, including platform design, cell culture method, real-time cytotoxic assay setup, and image-based analysis. A 2D experimental method is used for comparison, which effectively demonstrates the advantages of 3D microfluidic platforms in closely recapitulating immune responses within the tumor microenvironment. Moreover, a wide range of experimental possibilities and applications using microfluidic 3D cytotoxic assays is introduced in this chapter, along with their capabilities, limitations, and future outlook.

On-chip flow cytometer using integrated photonics for the detection of human leukocytes.

Differentiation between leukocyte subtypes like monocytes and lymphocytes is essential for cell therapy and research applications. To guarantee the cost-effective delivery of functional cells in cell therapies, billions of cells must be processed in a limited time. Yet, the sorting rates of commercial cell sorters are not high enough to reach the required yield. Process parallelization by using multiple instruments increases variability and production cost. A compact solution with higher throughput can be provided by multichannel flow cytometers combining fluidics and optics on-chip. In this work, we present a micro-flow cytometer with monolithically integrated photonics and fluidics and demonstrate that both the illumination of cells, as well as the collection of scattered light, can be realized using photonic integrated circuits. Our device is the first with sufficient resolution for the discrimination of lymphocytes and monocytes. Innovations in microfabrication have enabled complete integration of miniaturized photonic components and fluidics in a CMOS-compatible wafer stack. In combination with external optics, the device is ready for the collection of fluorescence using the on-chip excitation.

LymphoDose: a lymphocyte dose estimation framework-application to brain radiotherapy.

Objective. Severe radiation-induced lymphopenia occurs in 40% of patients treated for primary brain tumors and is an independent risk factor of poor survival outcomes. We developed anin-silicoframework that estimates the radiation doses received by lymphocytes during volumetric modulated arc therapy brain irradiation.Approach. We implemented a simulation consisting of two interconnected compartmental models describing the slow recirculation of lymphocytes between lymphoid organs (M1) and the bloodstream (M2). We used dosimetry data from 33 patients treated with chemo-radiation for glioblastoma to compare three cases of the model, corresponding to different physical and biological scenarios: (H1) lymphocytes circulation only in the bloodstream i.e. circulation inM2only; (H2) lymphocytes recirculation between lymphoid organs i.e. circulation inM1andM2interconnected; (H3) lymphocytes recirculation between lymphoid organs and deep-learning computed out-of-field (OOF) dose to head and neck (H&N) lymphoid structures. A sensitivity analysis of the model's parameters was also performed.Main results. For H1, H2 and H3 cases respectively, the irradiated fraction of lymphocytes was 99.8 ± 0.7%, 40.4 ± 10.2% et 97.6 ± 2.5%, and the average dose to irradiated pool was 309.9 ± 74.7 mGy, 52.6 ± 21.1 mGy and 265.6 ± 48.5 mGy. The recirculation process considered in the H2 case implied that irradiated lymphocytes were irradiated in the field only 1.58 ± 0.91 times on average after treatment. The OOF irradiation of H&N lymphoid structures considered in H3 was an important contribution to lymphocytes dose. In all cases, the estimated doses are low compared with lymphocytes radiosensitivity, and other mechanisms could explain high prevalence of RIL in patients with brain tumors.Significance. Our framework is the first to take into account OOF doses and recirculation in lymphocyte dose assessment during brain irradiation. Our results demonstrate the need to clarify the indirect effects of irradiation on lymphopenia, in order to potentiate the combination of radio-immunotherapy or the abscopal effect.

Sex differences orchestrated by androgens at single-cell resolution.

Sex differences in mammalian complex traits are prevalent and are intimately associated with androgens1-7. However, a molecular and cellular profile of sex differences and their modulation by androgens is still lacking. Here we constructed a high-dimensional single-cell transcriptomic atlas comprising over 2.3 million cells from 17 tissues in Mus musculus and explored the effects of sex and androgens on the molecular programs and cellular populations. In particular, we found that sex-biased immune gene expression and immune cell populations, such as group 2 innate lymphoid cells, were modulated by androgens. Integration with the UK Biobank dataset revealed potential cellular targets and risk gene enrichment in antigen presentation for sex-biased diseases. This study lays the groundwork for understanding the sex differences orchestrated by androgens and provides important evidence for targeting the androgen pathway as a broad therapeutic strategy for sex-biased diseases.

Dissection and integration of bursty transcriptional dynamics for complex systems.

RNA velocity estimation is a potentially powerful tool to reveal the directionality of transcriptional changes in single-cell RNA-sequencing data, but it lacks accuracy, absent advanced metabolic labeling techniques. We developed an approach, TopicVelo, that disentangles simultaneous, yet distinct, dynamics by using a probabilistic topic model, a highly interpretable form of latent space factorization, to infer cells and genes associated with individual processes, thereby capturing cellular pluripotency or multifaceted functionality. Focusing on process-associated cells and genes enables accurate estimation of process-specific velocities via a master equation for a transcriptional burst model accounting for intrinsic stochasticity. The method obtains a global transition matrix by leveraging cell topic weights to integrate process-specific signals. In challenging systems, this method accurately recovers complex transitions and terminal states, while our use of first-passage time analysis provides insights into transient transitions. These results expand the limits of RNA velocity, empowering future studies of cell fate and functional responses.

Depleting myeloid-biased haematopoietic stem cells rejuvenates aged immunity.

Ageing of the immune system is characterized by decreased lymphopoiesis and adaptive immunity, and increased inflammation and myeloid pathologies1,2. Age-related changes in populations of self-renewing haematopoietic stem cells (HSCs) are thought to underlie these phenomena3. During youth, HSCs with balanced output of lymphoid and myeloid cells (bal-HSCs) predominate over HSCs with myeloid-biased output (my-HSCs), thereby promoting the lymphopoiesis required for initiating adaptive immune responses, while limiting the production of myeloid cells, which can be pro-inflammatory4. Ageing is associated with increased proportions of my-HSCs, resulting in decreased lymphopoiesis and increased myelopoiesis3,5,6. Transfer of bal-HSCs results in abundant lymphoid and myeloid cells, a stable phenotype that is retained after secondary transfer; my-HSCs also retain their patterns of production after secondary transfer5. The origin and potential interconversion of these two subsets is still unclear. If they are separate subsets postnatally, it might be possible to reverse the ageing phenotype by eliminating my-HSCs in aged mice. Here we demonstrate that antibody-mediated depletion of my-HSCs in aged mice restores characteristic features of a more youthful immune system, including increasing common lymphocyte progenitors, naive T cells and B cells, while decreasing age-related markers of immune decline. Depletion of my-HSCs in aged mice improves primary and secondary adaptive immune responses to viral infection. These findings may have relevance to the understanding and intervention of diseases exacerbated or caused by dominance of the haematopoietic system by my-HSCs.

Automatic classification of acute lymphoblastic leukemia cells and lymphocyte subtypes based on a novel convolutional neural network.

Acute lymphoblastic leukemia (ALL) is a life-threatening disease that commonly affects children and is classified into three subtypes: L1, L2, and L3. Traditionally, ALL is diagnosed through morphological analysis, involving the examination of blood and bone marrow smears by pathologists. However, this manual process is time-consuming, laborious, and prone to errors. Moreover, the significant morphological similarity between ALL and various lymphocyte subtypes, such as normal, atypic, and reactive lymphocytes, further complicates the feature extraction and detection process. The aim of this study is to develop an accurate and efficient automatic system to distinguish ALL cells from these similar lymphocyte subtypes without the need for direct feature extraction. First, the contrast of microscopic images is enhanced using histogram equalization, which improves the visibility of important features. Next, a fuzzy C-means clustering algorithm is employed to segment cell nuclei, as they play a crucial role in ALL diagnosis. Finally, a novel convolutional neural network (CNN) with three convolutional layers is utilized to classify the segmented nuclei into six distinct classes. The CNN is trained on a labeled dataset, allowing it to learn the distinguishing features of each class. To evaluate the performance of the proposed model, quantitative metrics are employed, and a comparison is made with three well-known deep networks: VGG-16, DenseNet, and Xception. The results demonstrate that the proposed model outperforms these networks, achieving an approximate accuracy of 97%. Moreover, the model's performance surpasses that of other studies focused on 6-class classification in the context of ALL diagnosis. RESEARCH HIGHLIGHTS: Deep neural networks eliminate the requirement for feature extraction in ALL classification The proposed convolutional neural network achieves an impressive accuracy of approximately 97% in classifying six ALL and lymphocyte subtypes.

RABiT-III: an Automated Micronucleus Assay at a Non-Specialized Biodosimetry Facility.

Micronuclei, detected through the cytokinesis-block micronucleus assay, are valuable indicators of ionizing radiation exposure, especially in short-term lymphocyte cultures. The peripheral human blood lymphocyte assay is recognized as a prime candidate for automated biodosimetry. In a prior project at the Columbia University Center for Radiological Research, we automated this assay using the 96-well ANSI/SLAS microplate standard format and relied on established biotech robotic systems named Rapid Automated Biodosimetry Tool (RABiT). In this study, we present the application of a similar automated biotech setup at an external high-throughput facility (RABiT-III) to implement the same automated cytokinesis-block micronucleus assay. Specifically, we employed the Agilent BRAVO liquid-handling system and GE IN Cell Analyzer 6000 imaging system in conjunction with the PerkinElmer Columbus image data storage and analysis system. Notably, this analysis system features an embedded PhenoLOGIC machine learning module, simplifying the creation of cell classification algorithms for CBMN assay image analysis and enabling the generation of radiation dose-response curves. This investigation underscores the adaptability of the RABiT-II CBMN protocol to diverse RABiT-III biotech robotic platforms in non-specialized biodosimetry centers. Furthermore, it highlights the advantages of machine learning in rapidly developing algorithms crucial for the high-throughput automated analysis of RABiT-III images.

Flow rate resonance of actively deforming particles.

Lymphoid organs are unusual multicellular tissues: they are densely packed, but the lymphocytes trafficking through them are actively moving. We hypothesize that the intriguing ability of lymphocytes to avoid jamming and clogging is in part attributable to the dynamic shape changes that cells undergo when they move. In this work, we test this hypothesis by investigating an idealized system, namely, the flow of self-propelled, oscillating particles passing through a narrow constriction in two dimensions (2D), using numerical simulations. We found that deformation allows particles with these properties to flow through a narrow constriction in conditions when non-deformable particles would not be able to do so. Such a flowing state requires the amplitude and frequency of oscillations to exceed threshold values. Moreover, a resonance leading to the maximum flow rate was found when the oscillation frequency matched the natural frequency of the particle related to its elastic stiffness. To our knowledge, this phenomenon has not been described previously. Our findings could have important implications for understanding and controlling flow in a variety of systems in addition to lymphoid organs, such as granular flows subjected to vibration.

Multiple waves of fetal-derived immune cells constitute adult immune system.

It has been over three decades since Drs. Herzenberg and Herzenberg proposed the layered immune system hypothesis, suggesting that different types of stem cells with distinct hematopoietic potential produce specific immune cells. This layering of immune system development is now supported by recent studies showing the presence of fetal-derived immune cells that function in adults. It has been shown that various immune cells arise at different embryonic ages via multiple waves of hematopoiesis from special endothelial cells (ECs), referred to as hemogenic ECs. However, it remains unknown whether these fetal-derived immune cells are produced by hematopoietic stem cells (HSCs) during the fetal to neonatal period. To address this question, many advanced tools have been used, including lineage-tracing mouse models, cellular barcoding techniques, clonal assays, and transplantation assays at the single-cell level. In this review, we will review the history of the search for the origins of HSCs, B-1a progenitors, and mast cells in the mouse embryo. HSCs can produce both B-1a and mast cells within a very limited time window, and this ability declines after embryonic day (E) 14.5. Furthermore, the latest data have revealed that HSC-independent adaptive immune cells exist in adult mice, which implies more complicated developmental pathways of immune cells. We propose revised road maps of immune cell development.

C-reactive protein, but not neutrophil-lymphocyte ratio, is inversely associated with muscle strength only in older men: NHANES 1999-2002.

To evaluate the association of inflammation (C-reactive protein (CRP) and neutrophil-lymphocyte ratio (NLR) levels) with muscle strength in older adults. We also aimed to evaluate whether these associations are sex-specific. A cross-sectional study was performed with data from the National Health and Nutrition Examination Survey (NHANES) 1999-2000 and 2001-2002. A total of 2387 individuals over 50 years of both sexes were evaluated, according to the eligibility criteria for the strength test. Muscle strength was measured by Kinetic Communicator isokinetic dynamometer; while the NLR was obtained by the ratio of the total neutrophil for lymphocyte count and CRP was quantified by latex nephelometry. Linear regression analyses, crude and adjusted for confounders, were used to estimate the coefficients and 95 % confidence intervals for peak strength (muscle strength) by tertiles of NLR and CRP. There was no association between NLR and peak strength for both sexes. CRP levels were inversely associated with peak force in men [2nd tertile ß = -3.33 (-15.92; 9.25); 3rd tertile ß = -24.69 (-41.18; -8.20), p for trend = 0.005], but not in women [2nd tertile ß = -3.22 (-15.00; 8.56); 3rd tertile ß = -9.23 (-28.40; -9.94), p for trend = 0.332]. In conclusion, NLR levels were not associated with muscle strength in both sexes. CRP levels were inversely associated with muscle strength in older men, but not in women, suggesting that the association between inflammation and muscle strength in older adults can be sex-specific.

Inulin fibre promotes microbiota-derived bile acids and type 2 inflammation.

Dietary fibres can exert beneficial anti-inflammatory effects through microbially fermented short-chain fatty acid metabolites^1,2, although the immunoregulatory roles of most fibre diets and their microbiota-derived metabolites remain poorly defined. Here, using microbial sequencing and untargeted metabolomics, we show that a diet of inulin fibre alters the composition of the mouse microbiota and the levels of microbiota-derived metabolites, notably bile acids. This metabolomic shift is associated with type 2 inflammation in the intestine and lungs, characterized by IL-33 production, activation of group 2 innate lymphoid cells and eosinophilia. Delivery of cholic acid mimics inulin-induced type 2 inflammation, whereas deletion of the bile acid receptor farnesoid X receptor diminishes the effects of inulin. The effects of inulin are microbiota dependent and were reproduced in mice colonized with human-derived microbiota. Furthermore, genetic deletion of a bile-acid-metabolizing enzyme in one bacterial species abolishes the ability of inulin to trigger type 2 inflammation. Finally, we demonstrate that inulin enhances allergen- and helminth-induced type 2 inflammation. Taken together, these data reveal that dietary inulin fibre triggers microbiota-derived cholic acid and type 2 inflammation at barrier surfaces with implications for understanding the pathophysiology of allergic inflammation, tissue protection and host defence.

Diverse mutational landscapes in human lymphocytes.

The lymphocyte genome is prone to many threats, including programmed mutation during differentiation1, antigen-driven proliferation and residency in diverse microenvironments. Here, after developing protocols for expansion of single-cell lymphocyte cultures, we sequenced whole genomes from 717 normal naive and memory B and T cells and haematopoietic stem cells. All lymphocyte subsets carried more point mutations and structural variants than haematopoietic stem cells, with higher burdens in memory cells than in naive cells, and with T cells accumulating mutations at a higher rate throughout life. Off-target effects of immunological diversification accounted for approximately half of the additional differentiation-associated mutations in lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every on-target IGHV mutation during the germinal centre reaction. Structural variation was 16-fold higher in lymphocytes than in stem cells, with around 15% of deletions being attributable to off-target recombinase-activating gene activity. DNA damage from ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory cells. The mutation burden and signatures of normal B cells were broadly similar to those seen in many B-cell cancers, suggesting that malignant transformation of lymphocytes arises from the same mutational processes that are active across normal ontogeny. The mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments.

ZBTB46 defines and regulates ILC3s that protect the intestine.

RORγt is a lineage-specifying transcription factor that is expressed by immune cells that are enriched in the gastrointestinal tract and promote immunity, inflammation and tissue homeostasis1-15. However, fundamental questions remain with regard to the cellular heterogeneity among these cell types, the mechanisms that control protective versus inflammatory properties and their functional redundancy. Here we define all RORγt+ immune cells in the intestine at single-cell resolution and identify a subset of group 3 innate lymphoid cells (ILC3s) that expresses ZBTB46, a transcription factor specifying conventional dendritic cells16-20. ZBTB46 is robustly expressed by CCR6+ lymphoid-tissue-inducer-like ILC3s that are developmentally and phenotypically distinct from conventional dendritic cells, and its expression is imprinted by RORγt, fine-tuned by microbiota-derived signals and increased by pro-inflammatory cytokines. ZBTB46 restrains the inflammatory properties of ILC3s, including the OX40L-dependent expansion of T helper 17 cells and the exacerbated intestinal inflammation that occurs after enteric infection. Finally, ZBTB46+ ILC3s are a major source of IL-22, and selective depletion of this population renders mice susceptible to enteric infection and associated intestinal inflammation. These results show that ZBTB46 is a transcription factor that is shared between conventional dendritic cells and ILC3s, and identify a cell-intrinsic function for ZBTB46 in restraining the pro-inflammatory properties of ILC3s and a non-redundant role for ZBTB46+ ILC3s in orchestrating intestinal health.

Ablation of cDC2 development by triple mutations within the Zeb2 enhancer.

The divergence of the common dendritic cell progenitor1-3 (CDP) into the conventional type 1 and type 2 dendritic cell (cDC1 and cDC2, respectively) lineages4,5 is poorly understood. Some transcription factors act in the commitment of already specified progenitors-such as BATF3, which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer4,6-but the mechanisms controlling the initial divergence of CDPs remain unknown. Here we report the transcriptional basis of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis7 suggested that Nfil3 acts upstream of Id2, Batf3 and Zeb2 in cDC1 development but did not reveal its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and chromatin immunoprecipitation followed by sequencing identified endogenous NFIL3 binding in the -165 kb Zeb2 enhancer8 at three sites that also bind the CCAAT-enhancer-binding proteins C/EBPα and C/EBPß. In vivo mutational analysis using CRISPR-Cas9 targeting showed that these NFIL3-C/EBP sites are functionally redundant, with C/EBPs supporting and NFIL3 repressing Zeb2 expression at these sites. A triple mutation of all three NFIL3-C/EBP sites ablated Zeb2 expression in myeloid, but not lymphoid progenitors, causing the complete loss of pre-cDC2 specification and mature cDC2 development in vivo. These mice did not generate T helper 2 (TH2) cell responses against Heligmosomoides polygyrus infection, consistent with cDC2 supporting TH2 responses to helminths9-11. Thus, CDP divergence into cDC1 or cDC2 is controlled by competition between NFIL3 and C/EBPs at the -165 kb Zeb2 enhancer.

Brain motor and fear circuits regulate leukocytes during acute stress.

The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On the one hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.

High neutrophil-to-lymphocyte ratio is a significant predictor of depressive symptoms in maintenance hemodialysis patients: a cross-sectional study.

BACKGROUND: Depression is one of the most important psychiatric disorders in chronic kidney disease patients who undergo maintenance hemodialysis (MHD). Previous studies have shown that low-grade inflammation is involved in the progression of depressive symptoms. The neutrophil-to-lymphocyte ratio (NLR) is an inflammatory marker that is inexpensive and easy to measure. However, the association between NLR and depression symptoms in MHD patients has not been examined. METHODS: In this single-center, cross-sectional study, we included 160 patients undergoing MHD. The Patient Health Questionnaire-9 (PHQ-9) was used to assess depressive symptoms. NLR was calculated as the ratio of neutrophils to lymphocytes. Multinomial logistic regression and multivariate linear regression analyses were used to examine the association between NLR and depressive symptoms in MHD patients. RESULTS: Depressive symptoms were detected in 36.7% of the 160 MHD patients. Multinomial logistic regression showed that NLR was a significant predictor of mild (odds ratio [OR]: 1.383, 95% confidence interval [CI]: 1.015-1.884, p = 0.04) and moderate/moderately severe depressive symptoms (OR: 1.441, 95% CI: 1.017-2.042, p = 0.04) in MHD patients, adjusted for age, sex, Kt/V, dialysis duration, history of kidney transplantation, history of hypertension, and Charlson comorbidity index score. In addition, multivariate linear regression analysis showed that NLR was an independent influencing factor for PHQ-9 score in MHD patients, after adjusting for confounding factors. CONCLUSIONS: These findings suggest that NLR can be used as a biomarker for predicting depressive symptoms in MHD patients.

KIT as a master regulator of the mast cell lineage.

The discovery in 1987/1988 and 1990 of the cell surface receptor KIT and its ligand, stem cell factor (SCF), was a critical achievement in efforts to understand the development and function of multiple distinct cell lineages. These include hematopoietic progenitors, melanocytes, germ cells, and mast cells, which all are significantly affected by loss-of-function mutations of KIT or SCF. Such mutations also influence the development and/or function of additional cells, including those in parts of the central nervous system and the interstitial cells of Cajal (which control gut motility). Many other cells can express KIT constitutively or during immune responses, including dendritic cells, eosinophils, type 2 innate lymphoid cells, and taste cells. Yet the biological importance of KIT in many of these cell types largely remains to be determined. We here review the history of work investigating mice with mutations affecting the white spotting locus (which encodes KIT) or the steel locus (which encodes SCF), focusing especially on the influence of such mutations on mast cells. We also briefly review efforts to target the KIT/SCF pathway with anti-SCF or anti-Kit antibodies in mouse models of allergic disorders, parasite immunity, or fibrosis in which mast cells are thought to play significant roles.

Neutrophil-to-lymphocyte ratio and lactate dehydrogenase for early diagnosis of AIDS patients with Talaromyces marneffei infection.

BACKGROUND: This study aimed to explore the value of neutrophil-to-lymphocyte ratio (NLR) in combination with routine blood tests, lactate dehydrogenase (LDH), and T-lymphocyte subsets for the early diagnosis of acquired immunodeficiency syndrome (AIDS) combined with Talaromyces marneffei (TM) infection. METHODS: A total of 166 confirmed AIDS patients were enrolled in this study. The observation group included 80 AIDS patients with TM infection, and the control group consisted of 86 AIDS patients with other complications. Regression analysis was performed to evaluate the predictive value of each index and the combination of these indexes for AIDS combined with TM infection using receiver operating characteristic (ROC) curve analysis. RESULTS: NLR and LDH were significantly higher in patients in the observation group compared with those in the control group, and the differences were statistically significant (P<0.05). There was no statistical difference in platelets, infantile granulocytes (IGM), and nucleated red blood cells (NRBC) between the 2 groups (P>0.05). The area under the operating characteristic curve (AUC) of the observed indicators were: NLR, 0.628; hemoglobin (HGB), 0.704; LDH, 0.607; lymphocyte (LYM) count, 0.744; CD4+ T lymphocyte count, 0.789; and CD8+ T lymphocyte count, 0.701. The combined AUC of multiple indicators was 0.815, with a sensitivity and specificity of 76.2% and 76.1%, respectively. CONCLUSIONS: NLR, HGB, LYM, LDH, and T lymphocyte subsets were diagnostic for early AIDS combined with TM infection , and CD4+ T lymphocytes had the best diagnostic efficacy alone.