Application of MIF in promoting intestinal plasma cell production and IgA production of patients with immune reconstruction insufficiency

Abstract

The invention discloses application of MIF in promoting intestinal plasma cell production and IgA production of patients with immune reconstitution insufficiency, and the research proves that expression reduction of CD74 on intestinal mucosa B cells of people with immune reconstitution failure is a reason causing intestinal IgA plasma cell production of people with immune reconstitution failure for the first time. And the CD74 agonist MIF is proved to be capable of increasing the generation of intestinal IgA plasma cells and IgA of people with immune reconstruction failure.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of MIF in promoting the production of intestinal plasma cells and IgA in patients with incomplete immune reconstitution. Background Art

[0002] AIDS, also known as acquired immunodeficiency syndrome (AIDS), is caused by infection with the human immunodeficiency virus (HIV) and is a global public health challenge. Effective antiretroviral therapy (ART) has significantly improved the prognosis for HIV-infected individuals, transforming HIV infection into a chronic disease and significantly reducing AIDS morbidity and mortality. With effective ART, most patients achieve virological suppression and immune recovery, known as immune responders (IRs). However, 15% to 30% of patients experience slow CD4+ T cell recovery and struggle to return to normal levels, even with effective ART. These individuals are known as immune non-responders (INRs). They face an increased risk of opportunistic infections, malignancies, and other complications, and have a poor prognosis. Currently, there are no reliable medications for early intervention, and the difficulty of early identification and the lack of interventions warrant special attention for this vulnerable population. The current definition is: In HIV-infected individuals receiving effective ART for at least four years, with a peripheral blood viral load below the limit of detection (<50 copies / mL) for at least three years, and a CD4+ T-cell count above 500 cells / µL are considered IRs, while those below 350 cells / µL are considered INRs. To date, there are no safe and effective intervention strategies to elevate and restore immune reconstitution to INRs.

[0003] Failure of immune reconstitution in HIV infection is multifactorial, and the precise mechanisms remain incompletely elucidated. However, persistent immune activation and inflammation are generally considered the primary drivers. The development of both AIDS and non-AIDS events is directly or indirectly the result of chronic inflammation, leading to AIDS being considered an inflammatory disease. During HIV infection, intestinal immune disturbances, microbial translocation, and intestinal damage are closely linked, contributing to the chronic inflammatory state seen in HIV-infected individuals and a key reason for the failure of CD4+ T cell recovery in individuals with high INRs. Imbalances in the gut immune-microbiome lead to intestinal barrier disruption, triggering translocation of intestinal microbiota and their metabolites into the circulation, continuously activating systemic immunity and driving chronic inflammation. Simultaneously, immune overactivation further suppresses CD4+ T cell production and exacerbates intestinal tissue damage. Significantly reduced intestinal mucosal IgA levels are a key factor contributing to immune reconstitution failure in HIV-infected individuals. HIV infection is often accompanied by a significant decrease in intestinal IgA levels, which leads to an imbalance in the gut microbiome and translocation of microbial products, triggering a systemic chronic inflammatory response. This accelerates the exhaustion of CD4+ T cells and hinders their recovery, becoming an important driving factor for the failure of immune reconstitution.

[0004] Currently, even during long-term antiretroviral therapy (ART) for HIV infection, CD4+ T cells may not return to normal levels. These individuals are known as immunological non-responders (INRs), or those with incomplete immune reconstitution. Currently, there is no effective treatment for incomplete immune reconstitution in HIV infection after ART.

[0005] While the "Traditional Chinese Medicine Preparation for Treating Incomplete Immune Reconstitution in HIV / AIDS and Its Preparation Method" (invention patent CN202410882407.5) and the "Kidney Bean Lectin Botanical Composition for Treating Immune Reconstitution Deficiency in HIV / AIDS and Its Preparation Method and Application" (invention patent CN201911052936.8) have demonstrated safety and promising results, the formulations employed are based on traditional Chinese medicine, requiring large dosages and requiring stable efficacy, making quality assurance and industrial production difficult. Furthermore, these formulations cannot maintain stability for extended periods after discontinuation of the drug. Furthermore, the intestinal tract of HIV patients is the first site of infection, the most severe site of infection, and the most difficult to recover from. These studies were based solely on assessments of patients' peripheral blood immune responses, which is a limitation. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present inventors pioneered the discovery that, compared with healthy controls (HCs) and individuals with successful immune reconstitution (IRs), the number of plasma cells in the intestinal mucosa of individuals with INRs was significantly reduced, while the number of B cells was significantly increased. Our team further discovered that in the intestinal mucosa of individuals with INRs, the differentiation of B cells into plasma cells was impaired, with CD74 expression levels on B cells significantly reduced, and this decrease was closely associated with a decrease in IgA plasma cell levels. Using CD74 agonists for stimulation, we found that intestinal IgA production was significantly increased in individuals with failed immune reconstitution.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides the use of CD74 or a CD74 agonist in the preparation of a medicament for promoting the production of intestinal plasma cells in patients with incomplete immune reconstitution.

[0009] Furthermore, the CD74 agonist includes MIF or its derivatives.

[0010] Furthermore, the patient's immune reconstitution deficiency is induced by HIV virus.

[0011] Furthermore, the plasma cells are IgA plasma cells.

[0012] Furthermore, the drug is administered orally, via suppository, around the lesion, on the lesion surface, topically, intravenously, parenterally, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally or subcutaneously.

[0013] Furthermore, the medicine also includes functional excipients.

[0014] Furthermore, the functional excipients include excipients, diluents, dispersing aids, suspending aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, and lubricants.

[0015] In some embodiments, specific examples of the functional excipients include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); proteins such as serum albumin, gelatin, or immunoglobulin; hydrophilic polyols; compounds such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0016] Furthermore, the drug is in the form of a pharmaceutically acceptable salt.

[0017] Furthermore, the pharmaceutically acceptable salts include acid addition salts or base addition salts.

[0018] Furthermore, acid addition salts include any one or a combination of at least two of hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, hexanoate, argininate, fumarate, nicotinate, phthalate or oxalate.

[0019] Furthermore, base addition salts include lithium salts, sodium salts, potassium salts, barium salts, calcium salts, magnesium salts, aluminum salts, ferric salts, ferrous salts, copper salts, zinc salts, or salts with morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histidine.

[0020] The term "pharmaceutically acceptable salt" as used herein includes conventional salts formed from pharmaceutically acceptable inorganic or organic acids or inorganic or organic bases, and acid addition salts of quaternary ammonium. More specific examples of suitable acid salts include salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succinic acid, glycolic acid, formic acid, lactic acid, maleic acid, tartaric acid, citric acid, pamoic acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthoic acid, hydroiodic acid, malic acid, tannic acid, etc. Other acids, such as oxalic acid, although not pharmaceutically acceptable per se, can be used to prepare salts used as intermediates to obtain compounds of the present invention and pharmaceutically acceptable salts thereof. More specific examples of suitable base salts include sodium, lithium, potassium, magnesium, aluminum, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine salts.

[0021] In some embodiments, when referring to a pharmaceutically acceptable salt of CD74 or a CD74 agonist, it generally means that it can be used in the pharmaceutical field, is not harmful to the product or mammals, or has a reasonable or acceptable benefit / risk ratio.

[0022] The term "agonist" as used herein is a key concept in pharmacology and biochemistry, referring to molecules or compounds that can bind to and activate specific receptors, thereby triggering a biological effect. Agonists mimic the effects of endogenous ligands (such as neurotransmitters and hormones) by binding to receptors, triggering cellular signaling pathways and ultimately producing physiological or pharmacological responses. Based on the degree of activation, agonists can be categorized as full agonists (producing a maximal effect) or partial agonists (producing only a partial effect). Agonists are structurally specific, requiring their molecular configuration to closely match the receptor binding site. This binding is typically achieved through hydrogen bonding, ionic bonding, or hydrophobic interactions. Mechanistically, agonists induce conformational changes in the receptor, which in turn activate G proteins, open ion channels, or initiate enzyme-linked reactions. For example, β-adrenergic receptor agonists activate adenylate cyclase via Gs proteins. Agonists are widely used in medicine, such as dopamine agonists for the treatment of Parkinson's disease and β2 receptor agonists for the relief of asthma. Their properties include affinity (binding capacity) and intrinsic activity (activation capacity), both of which are quantified using dose-response curves. Inverse agonists are a special type of drug that inhibits basal receptor activity. Agonist research involves receptor kinetics, structure-activity relationship analysis, and computer-aided drug design. High-throughput screening is often used in modern drug development to discover novel agonists. However, it is important to note that agonists may induce regulatory phenomena such as receptor desensitization or internalization, and long-term use requires consideration of tolerability.

[0023] The present invention provides the use of CD74 or a CD74 agonist in the preparation of a medicament for promoting IgA production.

[0024] Furthermore, the CD74 agonist includes MIF or its derivatives.

[0025] Furthermore, the patient's immune reconstitution deficiency is induced by HIV virus.

[0026] Furthermore, the drug is administered orally, via suppository, around the lesion, on the lesion surface, topically, intravenously, parenterally, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally or subcutaneously.

[0027] Furthermore, the IgA production is carried out by plasma cells.

[0028] Furthermore, the medicine also includes functional excipients.

[0029] Furthermore, the functional excipients include excipients, diluents, dispersing aids, suspending aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, and lubricants.

[0030] Furthermore, the drug is in the form of a pharmaceutically acceptable salt.

[0031] Furthermore, the pharmaceutically acceptable salts include acid addition salts or base addition salts.

[0032] Furthermore, acid addition salts include any one or a combination of at least two of hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, hexanoate, argininate, fumarate, nicotinate, phthalate or oxalate.

[0033] Furthermore, base addition salts include lithium salts, sodium salts, potassium salts, barium salts, calcium salts, magnesium salts, aluminum salts, ferric salts, ferrous salts, copper salts, zinc salts, or salts with morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histidine.

[0034] The present invention provides a drug comprising an effective amount of CD74 or a CD74 agonist.

[0035] In some embodiments, the term "effective dose" encompasses therapeutic, augmenting, alleviating, and prophylactic effective doses. It refers to a core concept in pharmacology, referring to the dose of a drug that produces a predetermined therapeutic effect (e.g., 50% maximal effect) in a specific population. Its key characteristics include: 1) a quantitative parameter for the dose-effect relationship; 2) a therapeutic index (TI = TD50 / ED50) that, together with the toxic dose (TD); and 3) regulation by factors such as bioavailability and receptor occupancy. Determination of the effective dose is a process throughout the drug lifecycle: preclinical studies establish initial predictions using in vitro EC50 and in vivo animal models; Phase I clinical trials define a safety margin based on the MTD and PK / PD models; and Phase II clinical trials utilize adaptive designs to optimize the ED90 (90% effective dose). Innovative technologies such as microdose CT / PET imaging can visualize drug distribution in target tissues in real time, assisting in precise dose selection.

[0036] The present invention provides a method for in vitro screening of drugs for enhancing the production of intestinal plasma cells of CD74-treated patients with incomplete immune reconstitution, comprising the following steps:

[0037] The intestinal secretory tissue of patients with incomplete in vitro immune reconstitution with or without the addition of CD74 was used as the drug action target, and the drugs to be screened were added to select drugs that can enhance the production of intestinal plasma cells in the group with the addition of CD74 but have no effect on the production of intestinal plasma cells in the group without the addition of CD74.

[0038] Furthermore, the plasma cells are IgA plasma cells.

[0039] CD74, the invariant chain of the major histocompatibility complex class II (MHC Class II), used in this invention, is a key transmembrane protein in the immune system, encoded by chromosome 5, and plays a central role in antigen presentation and immune regulation. 12 Its structural features include an N-terminal cytoplasmic region, a transmembrane region, and a C-terminal extracellular region, forming a functional trimeric structure on the cell surface. This conformation is crucial for binding to MHC Class II molecules.

[0040] The present invention provides the use of CD74 in screening drugs for enhancing CD74 to treat intestinal plasma cell production in patients with incomplete immune reconstitution.

[0041] Furthermore, the immune reconstitution is not entirely induced by HIV virus.

[0042] Furthermore, the plasma cells are IgA plasma cells.

[0043] The present invention provides a method for promoting the production of plasma cells in intestinal tissue of patients with incomplete immune reconstitution in vitro, which comprises using an effective amount of CD74 or a CD74 agonist.

[0044] Furthermore, the incomplete immune reconstitution is induced by HIV virus.

[0045] Furthermore, the plasma cells are IgA plasma cells.

[0046] In some embodiments, the intestinal tissue is from a human or non-human mammal. In some embodiments, non-human mammals include birds and non-human mammals, such as non-human primates, companion animals (such as dogs and cats), livestock (such as pigs, sheep, cows), and non-domesticated animals such as large cats. Regardless of the stage in the life cycle of the organism, the intestinal tissue of the present invention can be obtained from a human or non-human mammal.

[0047] Advantages and beneficial effects of the present invention:

[0048] This study confirmed for the first time that the reduced expression of CD74 on intestinal mucosal B cells in people with immune reconstitution failure is the cause of the production of intestinal IgA plasma cells in people with immune reconstitution failure, and confirmed that the CD74 agonist MIF can increase the production of intestinal IgA plasma cells and IgA in people with immune reconstitution failure.

[0049] Specifically, in the intestinal mucosa of INRs, the decreased expression of CD74 on B cells downregulated the level of XBP1 in intestinal mucosal B cells. XBP1 is a key factor for the differentiation of B cells into plasma cells. The downregulation of CD74 causes a decrease in the level of IgA plasma cells in the intestinal mucosa of INRs. The CD74 agonist MIF can increase the level of XBP1 in B cells, thereby increasing the production of IgA plasma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a graph of single-cell sequencing analysis results.

[0051] Figure 2 This is the reverse sequence analysis result diagram.

[0052] Figure 3 This is the patent factor analysis result chart.

[0053] Figure 4 The graph shows the results of IgA plasma cell levels in isolated intestinal cells without or with the addition of MIF.

[0054] Figure 5 The graph shows the results of XBP1 levels in isolated intestinal cells without or with the addition of MIF. DETAILED DESCRIPTION

[0055] The present invention will be further described below with reference to specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. The main features of the present invention may be applied to various embodiments without departing from the scope of the present invention.

[0056] Example 1

[0057] 1. Experimental Materials

[0058] (1) This study recruited chronic HIV-1 infected individuals who had received cART treatment for more than 2.5 years.

[0059] (2) Collection and processing of intestinal tissue

[0060] (3) Single-cell RNA sequencing: Intestinal mucosal cell suspensions were prepared using the protocol provided with the 10X Chromium 3' v3.1 kit. Library preparation and sequencing were provided by Shanghai Biotechnology Corporation (Shanghai, China). Sequencing was performed on the NovaSeq 6000 platform (Illumina, Inc., San Diego, CA).

[0061] 2. Experimental methods

[0062] 1. Preparation

[0063] (1) Preheat the constant temperature water bath to 37°C;

[0064] (2) Prepare crushed ice or ice box;

[0065] (3) Prepare complete culture medium: RPMI 1640 medium containing 10% fetal bovine serum, preheated at 37°C;

[0066] (4) Prepare 10 mL of 0.1% collagenase digestion solution: weigh 10 mg of collagenase and dissolve it in 10 mL of complete culture medium. After complete dissolution, filter through a 0.22 μm filter and preheat at 37°C.

[0067] (5) 1× DPBS pre-cooled at 4°C;

[0068] (6) Prepare washing solution and suspension: 1× DPBS solution containing 2% fetal bovine serum, pre-cooled at 4°C;

[0069] (7) 1× red blood cell lysis buffer pre-cooled at 4°C;

[0070] 2. Cell Suspension Preparation

[0071] (1) Carefully remove the tissue from the tissue preservation solution, place it in a sterile culture dish, and add 10 mL of 4°C pre-cooled 1× DPBS to rinse. Repeat 1-2 times to remove the residual tissue preservation solution;

[0072] (2) Place the rinsed tissue in a new culture dish, add a little digestive fluid and cut into pieces of about 1-2 mm 3 small pieces;

[0073] (3) Transfer the cut tissue to a 50 mL centrifuge tube containing 5 mL of digestion solution and place it in a 37°C water bath for shaking digestion. Gently shake the digestion solution every 5 minutes, and do not shake it violently;

[0074] (4) After digestion, pass the digestion solution through a pre-wetted 70 μm cell sieve and collect the filtrate into a new 50 mL centrifuge tube;

[0075] (5) Wash the cell sieve with 10 mL of 1× DPBS (containing 2% fetal bovine serum) and collect the filtrate into the same centrifuge tube;

[0076] (6) Centrifuge at 400 × g for 10 minutes at room temperature and discard the supernatant;

[0077] (7) Add 5 mL of 1× DPBS (containing 2% fetal bovine serum), gently pipette to mix, and resuspend the cells;

[0078] (8) Centrifuge at 300 × g for 5 minutes at room temperature and discard the supernatant;

[0079] (9) Add 3 mL of pre-chilled 1× red blood cell lysis buffer to the cell pellet, gently pipette to mix, do not vortex, and incubate at room temperature for 3 minutes;

[0080] (10) Centrifuge at 300 × g for 5 minutes at room temperature and discard the supernatant;

[0081] (11) Add 5 mL of pre-cooled 1× DPBS containing 2% fetal bovine serum and gently pipette to mix;

[0082] (12) Centrifuge at 300 × g for 5 minutes at room temperature and discard the supernatant;

[0083] (13) Add an appropriate amount of 1× DPBS (containing 2% FBS) and gently pipette to mix. Place on ice and prepare for counting.

[0084] 3. Cell Counting and Cell Viability Determination

[0085] (1) For cell counting, use the Countess® II automated cell counter.

[0086] (2) Gently pipette the cell suspension, take 10µL of cells and 10µL of 0.4% trypan blue and gently mix them evenly. Take 10µL and quickly add it to the Countess® II cell counting chamber. The results are shown in Table 1.

[0087] Table 1

[0088]

[0089] Note: Single cell suspension quality control requirements: (1) total cell count of 20,000-100,000 cells; (2) viability > 80%. Cells below 80% are not recommended for use in the instrument; (3) no adhesion between cells (aggregation rate < 5%); (4) no cell fragments or other particles larger than 40 μm; (5) no reverse transcription inhibitors and non-cellular nucleic acid molecules.

[0090] 4. Single-cell sequencing experiments

[0091] 10 ×Genomics machine and reverse transcription

[0092] (1) The prepared cell suspension, 10× barcoded gel beads, and oil were added to different chambers of Chromium ChipG, respectively, to form gel beads (GEM) via the 10× Genomics Chromium system.

[0093] (2) The GEM is transferred into a PCR instrument for reverse transcription. The reverse transcription primer containing 30nt oligo-dT on the gel magnetic beads causes the poly-A RNA in the cells to be reverse transcribed into a single strand of cDNA with barcode and UMI information;

[0094] (3) Purification of single-strand cDNA using magnetic beads;

[0095] (4) PCR amplification of the purified cDNA;

[0096] (5) The concentration of cDNA was determined using a Qubit nucleic acid quantifier, and the fragment size was determined using an Agilent 2100 bioanalyzer.

[0097] 5. Sequencing Library Construction

[0098] (1) After cDNA amplification, enzyme digestion and fragmentation are completed, and the optimal fragments are screened by magnetic beads. End repair, A addition, and adapter connection to Read2 sequencing primers are performed, and then a cDNA library containing P5 and P7 adapters is constructed by PCR.

[0099] (2) The library is purified using magnetic beads;

[0100] (3) The library concentration was detected using Qubit and the fragment size was detected using Agilent 4200 bioanalyzer.

[0101] Sequencing on the machine

[0102] Cluster generation and first-direction sequencing primer hybridization were completed according to the Illumina instructions. Cells harboring clusters were loaded onto the sequencing instrument. Paired-end sequencing was performed using the paired-end sequencing protocol. The sequencing process was controlled by Illumina's data collection software, which provided real-time data analysis.

[0103] 6. Single-cell Data Analysis

[0104] (1) Raw sequencing data processing

[0105] Cell Ranger v6.0 single-cell transcriptome sequencing data processing software was used to transfer single-cell data from Fastq files to cell expression matrices. The raw data was subjected to data quality control filtering, alignment, quantification, identification, and recovered cell identification to ultimately obtain the gene expression matrix of each cell. The details are as follows: (1) Cell barcode and UMI extraction; (2) Sequence information obtained from each sample was aligned with the GRCh38 human genome; (3) Cell-specific identification code (Barcode) correction: The Barcode sequence information obtained by sequencing was aligned with the known Barcode sequence in the database. The barcode sequence (measured sequence) that is completely consistent with the known Barcode in the database is the true sequence. (4) Specific molecular label (UMI) filtering and correction; (5) UMI counting;

[0106] For multi-sample integration, the top 2000 most variable genes were first identified. For each sample, the top 2000 most variable genes were identified based on the mean and dispersion (variance / mean) of all genes for subsequent integration analysis.

[0107] Data quality control and normalization

[0108] Using Seurat 4.0 to import expression profiles generated by Cell Ranger, we can calculate not only the number of genes and UMIs per cell, but also the expression of mitochondrial genes per cell. Typically, we filter out cells with fewer than 500 genes, less than 1000 UMIs, and a mitochondrial fraction exceeding 15%. Next, we use Scrublet software to remove doublets and avoid cases where multiple cells have high UMI expression.

[0109] (2) Cell clustering and visualization

[0110] A graph-based clustering algorithm was used to perform cluster analysis on the normalized data, and the Uniform Manifold Approximation and Projection (UMAP) algorithm was used for data visualization.

[0111] (3) Marker gene analysis

[0112] The Wilcoxon algorithm was used to analyze the signature genes of all clusters, and the signature genes were scored by comparing one group to the rest. Genes with high expression in each cluster, logFC > 0.25, and expression in at least 20% of cells were selected as the significant signature genes of the cluster.

[0113] (4) Cell type annotation

[0114] Cell types were annotated using the single-cell sequencing cell type annotation software SingleR in combination with signature genes. SingleR uses an RNAseq database as a reference, selects genes that are highly variable across cell types in the reference database, and then calculates the correlation between the predicted cell type and the reference database. This correlation is then recursively calculated by removing the cell types with the lowest correlation, ultimately yielding the predicted cell type annotation.

[0115] 7. Statistical Analysis

[0116] Statistical analyses were performed using R Studio 4.1.0, GraphPad Prism 8.0, and SPSS 26.0. Mann-Whitney and Kruskal-Wallis nonparametric tests were used to compare results between two groups and multiple groups, respectively. Spearman's rank correlation test was used to determine correlations between variables. Statistically significant differences were set at P < 0.05.

[0117] 3. Experimental results

[0118] like Figure 1 As shown, single-cell sequencing analysis showed that INRs intestinal B cells and TFH interact with the MIF / CD74 receptor ligand pair. Figure 2 As shown in Figure 2, reverse time series analysis revealed that there was a disorder of B cell differentiation into plasma cells in the population with INRs. Figure 3 As shown, transcription factor analysis found that XBP1, a key transcription factor for B cell differentiation into plasma cells, was significantly reduced in INRs populations, and the level of XBP1 in B cells was positively correlated with the level of intestinal IgA.

[0119] Example 2

[0120] 1. Experimental methods 1. Cell Suspension Preparation (1) Carefully remove the tissue from the tissue preservation solution, place it in a sterile culture dish, and add 10 mL of 4°C pre-cooled 1× DPBS to rinse. Repeat 1-2 times to remove the residual tissue preservation solution; (2) Place the rinsed tissue in a new culture dish, add a little digestive fluid and cut into pieces of about 1-2 mm 3 small pieces; (3) Transfer the cut tissue to a 50 mL centrifuge tube containing 5 mL of digestion solution and place it in a 37°C water bath for shaking digestion. Gently shake the digestion solution every 5 minutes, and do not shake it violently; (4) After digestion, pass the digestion solution through a pre-wetted 70 μm cell sieve and collect the filtrate into a new 50 mL centrifuge tube; (5) Wash the cell sieve with 10 mL of 1× DPBS (containing 2% fetal bovine serum) and collect the filtrate into the same centrifuge tube; (6) Centrifuge at 400 × g for 10 minutes at room temperature and discard the supernatant; (7) Add 5 mL of 1× DPBS (containing 2% fetal bovine serum), gently pipette to mix, and resuspend the cells; (8) Centrifuge at 300 × g for 5 minutes at room temperature and discard the supernatant; (9) Add 3 mL of pre-chilled 1× red blood cell lysis buffer to the cell pellet, gently pipette to mix, do not vortex, and incubate at room temperature for 3 minutes; (10) Centrifuge at 300 × g for 5 minutes at room temperature and discard the supernatant; (11) Add 5 mL of pre-cooled 1× DPBS containing 2% fetal bovine serum and gently pipette to mix; (12) Centrifuge at 300 × g for 5 minutes at room temperature and discard the supernatant; (13) Add an appropriate amount of 1× DPBS (containing 2% FBS) and gently pipette to mix. Place on ice and prepare for counting.

[0121] 2. Cell Counting and Cell Viability Determination (1) For cell counting, use the Countess® II automated cell counter. (2) Gently pipette the cell suspension, take 10µL of cells and 10µL of 0.4% trypan blue and gently mix them evenly. Take 10µL and quickly add it to the Countess® II cell counting chamber. The specific measurement results are shown in Table 2.

[0122] Table 2

[0123]

[0124] Note: Single cell suspension quality control requirements: (1) total cell count of 20,000-100,000 cells; (2) viability > 80%. Cells below 80% are not recommended for use in the instrument; (3) no adhesion between cells (aggregation rate < 5%); (4) no cell fragments or other particles larger than 40 μm; (5) no reverse transcription inhibitors and non-cellular nucleic acid molecules.

[0125] CD4 cells and B cells were purified by flow cytometry, and the levels of IgA plasma cells were detected after MIF stimulation and co-culture for 7 days.

[0126] 2. Experimental results

[0127] like Figure 4 As shown in the figure, the levels of IgA plasma cells and IgA in the supernatant of the isolated intestinal cells increased in the experimental group with MIF compared to the control group without MIF. Figure 5 As shown, the production of XBP1 in isolated intestinal cells was significantly increased in the experimental group with MIF addition compared with the control group without MIF addition.

[0128] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. Use of CD74 or CD74 agonists in the preparation of drugs that promote the production of intestinal plasma cells in patients with incomplete immune reconstitution.

2. The use according to claim 1, wherein the CD74 agonist comprises MIF or a derivative thereof; Preferably, the patient's immune reconstitution deficiency is induced by HIV virus.

3. The use according to claim 1, wherein the drug is administered orally, via suppository, around the lesion, on the lesion surface, topically, intravenously, parenterally, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally or subcutaneously; Preferably, the drug further comprises a functional excipient; Preferably, the functional excipients include excipients, diluents, dispersing aids, suspending aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, and lubricants; Preferably, the drug is in the form of a pharmaceutically acceptable salt; Preferably, the pharmaceutically acceptable salt includes an acid addition salt or a base addition salt; Preferably, the acid addition salt comprises any one or a combination of at least two of hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, hexanoate, argininate, fumarate, nicotinate, phthalate or oxalate; Preferably, the base addition salt includes a lithium salt, a sodium salt, a potassium salt, a barium salt, a calcium salt, a magnesium salt, an aluminum salt, a ferric salt, a ferrous salt, a copper salt, a zinc salt, or a salt with morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histidine.

4. Use of CD74 or CD74 agonists in the preparation of drugs for promoting IgA production.

5. The use according to claim 4, wherein the CD74 agonist comprises MIF or its derivatives; Preferably, the patient's immune reconstitution deficiency is induced by HIV virus.

6. The use according to claim 4, wherein the drug is administered orally, via suppository, around the lesion, on the lesion surface, topically, intravenously, parenterally, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally or subcutaneously; Preferably, the drug further comprises a functional excipient; Preferably, the functional excipients include excipients, diluents, dispersing aids, suspending aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, and lubricants; Preferably, the drug is in the form of a pharmaceutically acceptable salt; Preferably, the pharmaceutically acceptable salt includes an acid addition salt or a base addition salt; Preferably, the acid addition salt comprises any one or a combination of at least two of hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, hexanoate, argininate, fumarate, nicotinate, phthalate or oxalate; Preferably, the base addition salt includes a lithium salt, a sodium salt, a potassium salt, a barium salt, a calcium salt, a magnesium salt, an aluminum salt, a ferric salt, a ferrous salt, a copper salt, a zinc salt, or a salt with morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histidine.

7. A medicament comprising an effective amount of CD74 or an agonist of CD74.

8. A method for in vitro screening of drugs that enhance CD74 production in intestinal plasma cells for treating patients with incomplete immune reconstitution, comprising the following steps: The intestinal secretory tissue of patients with incomplete in vitro immune reconstitution with or without the addition of CD74 was used as the drug action target, and the drugs to be screened were added to select drugs that can enhance the production of intestinal plasma cells in the group with the addition of CD74 but have no effect on the production of intestinal plasma cells in the group without the addition of CD74.

9. Application of CD74 in screening drugs that enhance CD74 production in intestinal plasma cells for the treatment of patients with incomplete immune reconstitution; Preferably, the immune reconstitution is not entirely HIV-induced.

10. A method for promoting the production of plasma cells in intestinal tissue of patients with incomplete immune reconstitution in vitro, the method comprising administering an effective amount of CD74 or a CD74 agonist; Preferably, the incomplete immune reconstitution is induced by HIV virus.

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