Article CXCR3 Provides a Competitive Advantage for Retention of Mycobacterium tuberculosis-Specific Tissue-Resident Memory T Cells Following a Mucosal Tuberculosis Vaccine
文章 CXCR3 为黏膜结核疫苗后结核分枝杆菌特异性组织驻留记忆 T 细胞的保留提供竞争优势
2. Materials and Methods
2. 材料与方法
2.1. Mouse Strains
2.1. 小鼠品系
All mice had a C57Bl/6 (B6) background. Female WT mice were purchased from Australian Bio Resources (Moss Vale, NSW, Australia). B6.129P2-Cx cr 3 tm 1 D gen/J $({\bf C}{\bf{X}}{\bf{C}}{\bf{R}}3^{-/-})$ mice and B6.129S4-Cx cr 3 tm 1 A rsa/SoghJ (CiBER) mice were purchased from The Jackson Laboratory (Bar Harbour, ME, USA). Mice expressing transgenic T cell receptors specific for p25 (P25 mice) were kindly provided by Professor Joel Ernst (University of California, San Francisco, CA, USA). P25 mice were crossed with $\mathrm{CXCR}3^{-/-}$ mice in-house. All experiments involving mice were approved by the Sydney Local Health District Animal Welfare Committee under protocols 2013-075B and 2016-044. The animals were housed in the Centenary Institute Animal Facility and maintained under specific pathogen-free conditions.
所有小鼠均为C57Bl/6 (B6) 背景。雌性野生型 (WT) 小鼠购自Australian Bio Resources (澳大利亚新南威尔士州莫斯维尔)。B6.129P2-Cx cr 3 tm 1 D gen/J $({\bf C}{\bf{X}}{\bf{C}}{\bf{R}}3^{-/-})$ 小鼠和B6.129S4-Cx cr 3 tm 1 A rsa/SoghJ (CiBER) 小鼠购自The Jackson Laboratory (美国缅因州巴港)。特异性识别p25的转基因T细胞受体小鼠 (P25小鼠) 由Joel Ernst教授 (美国加利福尼亚大学旧金山分校) 惠赠。P25小鼠与本实验室培育的 $\mathrm{CXCR}3^{-/-}$ 小鼠进行杂交。所有动物实验均经悉尼地方卫生区动物福利委员会批准 (协议编号2013-075B和2016-044)。实验动物饲养于Centenary Institute动物设施，并在无特定病原体条件下维持。
2.2. rIAV Vaccine and Immunization
2.2. rIAV 疫苗与免疫接种
The recombinant H1N1 PR8.p25 virus was engineered to express the $\mathtt{p}25$ peptide (F QD AYN A AG GH NAV F) from M. tuberculosis Antigen $85\mathrm{B}_{240-255}$ ( $\mathrm{}85\mathrm{B},$ , Rv1886c) as previously described [34,35]. This peptide contains an immuno dominant, $\mathrm{IA}^{\flat}$ -restricted $\mathrm{CD4^{+}}$ T epitope recognised by P25 T cell receptor transgenic mice [36,37]. In brief, cDNA of each of the eight viral genomic segments was prepared, with the modified form of the NA gene incorporating the p25 peptide. These were trans fec ted into a co-culture of human embryonic kidney 293 (HEK293) cells and Madin–Darby canine kidney cells (MDCK). Viruses generated from this culture were collected and used to infect embryo nate d chicken eggs, and the rIAV was recovered from these eggs after $48\mathrm{{h}}$ . Viral titre was determined by plaque assay. Recombinant NA gene expression was confirmed by amplifying viral RNA by reverse transcriptase polymerase chain reaction (RT-PCR), followed by DNA sequencing.
重组H1N1 PR8.p25病毒经过改造，可表达来自结核分枝杆菌抗原85B240-255 (Ag85B, Rv1886c)的p25肽段(F QD AYN A AG GH NAV F)，如先前文献所述[34,35]。该肽段包含一个免疫显性、受IAb限制的CD4+ T细胞表位，能被P25 T细胞受体转基因小鼠识别[36,37]。简言之，我们制备了包含修饰版NA基因（整合p25肽段）的八段病毒基因组cDNA，将其转染至人胚胎肾293细胞(HEK293)与马-达二氏犬肾细胞(MDCK)的共培养体系。收集培养产生的病毒后接种鸡胚，48小时后从鸡胚中回收重组流感病毒(rIAV)。通过噬斑试验测定病毒滴度，采用逆转录聚合酶链反应(RT-PCR)扩增病毒RNA并经DNA测序确认重组NA基因的表达。
For immunization, mice were an aes thetis ed with $56~\mathrm{mg/kg}$ body weight ketamine and $7\mathrm{mg/kg}$ body weight xylazine, administered by intra peritoneal injection. Mice were immunized by the intranasal $(\mathrm{i}/\mathrm{n})$ route by delivering $50~\mathrm{L}$ PBS containing 20 pfu of PR8.p25 to their nostrils. From 3 days post-immunization (p.i.), mice were monitored and weighed daily until fully recovered.
免疫时，用56 mg/kg体重的氯胺酮和7 mg/kg体重的赛拉嗪对小鼠进行麻醉，通过腹腔注射给药。通过鼻内(i/n)途径免疫，向小鼠鼻孔滴注50 μL含20 pfu PR8.p25的PBS溶液。从免疫后第3天(p.i.)开始，每天监测小鼠体重直至完全恢复。
2.3. Preparation of Single-Cell Suspensions
2.3. 单细胞悬液的制备
Following sacrifice, the lungs were perfused through the left ventricle of the heart with $10\mathrm{mL}$ PBS, except when intra vascular staining (IVS) was performed to distinguish leukocytes in the lung parenchyma from those in the vas cula ture [38]. For IVS, mice were injected i.v. with $1\mathrm{g}$ anti-CD45-APC-Cy7 antibody in $200~{\mathrm{L}}$ PBS 3–5 min prior to euthanasia. Lungs were diced and incubated in $5\mathrm{mL}$ Roswell Park Memorial Institute media containing $10%$ fetal calf serum (cRPMI) with $50\mathrm{U}/\mathrm{mL}$ collagen as e type IV (Sigma) and $26~{\mathrm{g/mL}}$ DNAse type I (Sigma) for $30\mathrm{min}$ at $37^{\circ}C$ . Red blood cells were lysed in ACK lysis buffer and cells re suspended in cRPMI.
处死后，通过心脏左心室用10 mL PBS灌注肺部，除非进行血管内染色(IVS)以区分肺实质和脉管系统中的白细胞[38]。进行IVS时，在小鼠安乐死前3-5分钟静脉注射1 μg抗CD45-APC-Cy7抗体(溶于200 μL PBS)。将肺组织切碎后置于5 mL含10%胎牛血清的Roswell Park Memorial Institute培养基(cRPMI)中，加入50 U/mL IV型胶原酶(Sigma)和26 μg/mL I型DNA酶(Sigma)，37°C孵育30分钟。用ACK裂解液溶解红细胞后，将细胞重悬于cRPMI中。
2.4. Antibodies
2.4. 抗体
The antibodies and staining reagents used for cell staining in flow cytometry and for binding $\mathrm{IFN-}\gamma$ in enzyme-linked immunospot (ELISpot) and experiments are listed in Table 1.
流式细胞术细胞染色及酶联免疫斑点(ELISpot)实验中用于结合$\mathrm{IFN-}\gamma$的抗体和染色试剂列于表1。
Table 1. Monoclonal antibodies and staining reagents used in experiments.
| Assay | Marker | Fluorophore | Clone | Manufacturer |
| Flow Cytometry | CD3 | PerCP-Cy5.5 | 145-2C11 | BioLegend (San Diego, CA, USA) |
| CD3 | PE-Cy7 | 145-2C11 | BD Biosciences (Sydney, Australia) | |
| CD3 | N/A | 145-2C11 | BDBiosciences | |
| CD28 | N/A | 37.51 | BD Biosciences | |
| CD4 | AF700 | RM4-5 | BD Biosciences | |
| CD44 | FITC | IM7 | BDBiosciences | |
| CD45.1 | Biotin | A20 | BDBiosciences | |
| CD45.2 | PerCP-Cy5.5 | 104 | BioLegend | |
| CD62L | eF450 | MEL-14 | eBioscience (San Diego, CA, USA) | |
| CD69 | PE | H1.2F3 | BD Biosciences | |
| IFN- | PE | XMG1.2 | BD Biosciences | |
| IFN-Y | FITC | XMG1.2 | BDBiosciences | |
| TNF | APC | MP6-XT22 | BDBiosciences | |
| TNF | PE | MP6-XT22 | BioLegend | |
| IL-2 | APC | JES6-5H4 | BioLegend | |
| UV LIVE/DEAD? | UV LIVE/DEAD? | N/A | BioLegend | |
| Biotin | Pacific orange | N/A | Invitrogen (Waltham, MA, USA) | |
| ELISpot | p25 tetramer | APC | N/A | NIH Tetramer Core Facility |
| IFN-Y | N/A | AN18 | Produced in house | |
| IFN- | N/A | XMG1.2 | Produced in house |
表 1: 实验中使用的单克隆抗体和染色试剂。
| 检测方法 | 标记物 | 荧光染料 | 克隆号 | 生产商 |
|---|---|---|---|---|
| 流式细胞术 | CD3 | PerCP-Cy5.5 | 145-2C11 | BioLegend (San Diego, CA, USA) |
| CD3 | PE-Cy7 | 145-2C11 | BD Biosciences (Sydney, Australia) | |
| CD3 | N/A | 145-2C11 | BDBiosciences | |
| CD28 | N/A | 37.51 | BD Biosciences | |
| CD4 | AF700 | RM4-5 | BD Biosciences | |
| CD44 | FITC | IM7 | BDBiosciences | |
| CD45.1 | Biotin | A20 | BDBiosciences | |
| CD45.2 | PerCP-Cy5.5 | 104 | BioLegend | |
| CD62L | eF450 | MEL-14 | eBioscience (San Diego, CA, USA) | |
| CD69 | PE | H1.2F3 | BD Biosciences | |
| IFN- | PE | XMG1.2 | BD Biosciences | |
| IFN-Y | FITC | XMG1.2 | BDBiosciences | |
| TNF | APC | MP6-XT22 | BDBiosciences | |
| TNF | PE | MP6-XT22 | BioLegend | |
| IL-2 | APC | JES6-5H4 | BioLegend | |
| UV LIVE/DEAD? | UV LIVE/DEAD? | N/A | BioLegend | |
| Biotin | Pacific orange | N/A | Invitrogen (Waltham, MA, USA) | |
| ELISpot | p25 tetramer | APC | N/A | NIH Tetramer Core Facility |
| IFN-Y | N/A | AN18 | Produced in house | |
| IFN- | N/A | XMG1.2 | Produced in house |
2.5. Flow Cytometry and Intra cytoplasmic Cytokine Staining
2.5. 流式细胞术与胞内细胞因子染色
For antigen stimulation and Intra cytoplasmic Cytokine Staining (ICS) assays, $10^{6}$ cells from each suspension were cultured at $37^{\circ}C,$ $^{4\textrm{h}}$ in a U-bottom 96-well plate in $200~{\mathrm{L}}$ of cRPMI containing $10\mathrm{g/mL}$ Brefeldin A and either no stimulant (negative controls), $10\mathrm{g/mL}$ p25 peptide (GenScript; Piscataway, NJ, USA), or $50\mathrm{g/mL}$ anti-CD3 antibody and $25\mathrm{g/mL}$ anti-CD28 antibody (positive controls). Cells were re suspended in $40\mathrm{L}$ of cRPMI with $1%$ FCS and $10\mathrm{g/mL}$ of MHC $\mathrm{IA^{+}{-}p}25$ tetramer (NIH Tetramer Core Facility, Atlanta, GA, USA), and incubated at $37^{\circ}C$ for $^{1\textrm{h}}$ . Cells were incubated with Fc-block at $4^{\circ}C,$ for $20\mathrm{min}$ , then antibody staining was performed at $4^{\circ}C,$ $20\mathrm{min}$ . For experiments with bio tiny late d antibodies, cells were re suspended in $100~{\mathrm{L}}$ FACS buffer containing strep t avid in-conjugated flu or oph ore (Invitrogen) at $4^{\circ}\mathrm{C}.$ , $20\mathrm{min}$ . For ICS, cells were then suspended in $100\mathrm{L}$ Fix/Perm (BD) for $20\mathrm{min}$ at $4^{\circ}C$ . Cytokine staining was performed in $1\times$ Perm/Wash at $4^{\circ}C,$ 20 min (BD). Cells were re suspended in $100~{\mathrm{L}}$ of formalin and run on an LSR Fortessa (BD). Gating to identify ${\mathrm{CD}}3^{+}{\mathrm{CD}}4^{+}$ T lymphocytes (Figure S1A–D) and cytokine $^+\mathrm{CD}3^{+}\mathrm{CD}4^{+}$ T cells (Figure S2A–C) was performed using Flowjo version 9.8.2. Boolean gating was used to identify cells that expressed cytokines, singly or in combination.
在抗原刺激和胞内细胞因子染色(ICS)实验中，将每份悬浮液中的10^6个细胞置于U型底96孔板中，于37°C条件下培养4小时。培养体系为200 μL含10 μg/mL布雷菲德菌素A的cRPMI培养基，分别设置以下处理组：无刺激剂(阴性对照)、10 μg/mL p25多肽(GenScript公司)、或50 ng/mL抗CD3抗体+25 ng/mL抗CD28抗体(阳性对照)。细胞重悬于40 μL含1%胎牛血清和10 μg/mL MHC IA^+-p25四聚体(NIH四聚体核心设施)的cRPMI中，37°C孵育1小时。随后于4°C用Fc阻断剂预处理20分钟，再进行4°C抗体染色20分钟。使用生物素化抗体的实验中，细胞重悬于100 μL含链霉亲和素偶联荧光素的FACS缓冲液(Invitrogen)，4°C孵育20分钟。ICS实验时，细胞用100 μL Fix/Perm(BD)于4°C固定20分钟，随后在1×Perm/Wash(BD)中4°C避光进行细胞因子染色20分钟。最终细胞重悬于100 μL甲醛溶液，使用LSR Fortessa流式细胞仪(BD)检测。采用Flowjo 9.8.2软件进行设门分析：CD3^+CD4^+ T淋巴细胞(图S1A-D)及细胞因子^+CD3^+CD4^+ T细胞(图S2A-C)，并通过布尔逻辑门鉴定单独或共表达细胞因子的细胞亚群。
2.6. Adoptive Transfer
2.6. 过继性转移
For adoptive transfer experiments, sp leno cyte s were obtained from naïve $\mathrm{P}25\mathrm{RAG^{-/-}}$ mice that were either $\mathrm{CXC\hat{R}3^{w t/w t}}$ or $\dot{\mathrm{C}}{\times}{\mathrm{CR}3^{-}}/-$ and $\mathrm{CD4^{+}}$ T cells in the suspensions quantified by flow cytometry. Then, 25,000 P25 CD4+ T cells of each type were transferred by i.v. into B6 mice.
对于过继转移实验，脾淋巴细胞取自未致敏的$\mathrm{P}25\mathrm{RAG^{-/-}}$小鼠，这些小鼠分别为$\mathrm{CXC\hat{R}3^{w t/w t}}$或$\dot{\mathrm{C}}{\times}{\mathrm{CR}3^{-}}/-$基因型，并通过流式细胞术对悬浮液中的$\mathrm{CD4^{+}}$T细胞进行定量。随后，将每种类型的25,000个P25 CD4+ T细胞通过静脉注射转移至B6小鼠体内。
2.7. IFNγ ELISpot
2.7. IFNγ ELISpot
The frequency of $\mathrm{IFN-}\gamma$ -producing T cells in tissue samples was assessed by ELISpot. Multi Screen® 96-well filter plates (Merck; Rahway, NJ, USA) were wetted with $20~\mathrm{L}$ of $35%$ ethanol and washed with PBS. Anti-IFN $\gamma$ monoclonal antibody (mAb; clone AN18), $15\mathrm{g/mL}$ in PBS, was added to each well and incubated for $16\mathrm{h}$ at $4^{\circ}C$ . Wells were blocked with cRPMI $(2\mathrm{h},37^{\circ}\mathrm{C})$ and washed with PBS. $0.5\times10^{5}$ lung cells or $1\times10^{5}$ spleen cells were added to each well in the presence of $10\mathrm{g/mL}$ antigen (see specific experiments) or $5\mathrm{g/mL}$ con ca naval in A (Sigma; Melbourne, Australia) in cRPMI. Plates were incubated at $37^{\circ}C,$ $5%$ $\mathrm{CO}_{2}$ overnight. Wells were washed with PBS containing $0.01%$ Tween-20. Anti-IFN $\gamma$ -biotin mAb (clone XMG-1.2), $2.5~{\mathrm{g/mL}}$ in PBS, was added and incubated for $2\mathrm{h}$ at $37^{\circ}C$ . Then, the wells were washed as before and 1:1000 avidin–alkaline phosphatase (Sigma) in PBS added to each well and incubated for $45\mathrm{min}$ at room temperature, followed by washing as before. Next, $100~{\mathrm{L}}$ of AP substrate solution (Bio-Rad, Sydney, Australia) diluted in NPP buffer was added to each well and the plate was incubated until spots were visible, then washed with water. Spots were counted using an AID ELISpot reader (Melbourne, Australia) with the software ELISpot Reader version 6.0.
通过ELISpot检测组织样本中产生$\mathrm{IFN-}\gamma$的T细胞频率。使用Multi Screen® 96孔滤板(Merck; Rahway, NJ, USA)，每孔加入$20~\mathrm{L}$ $35%$乙醇润湿后PBS冲洗。每孔加入PBS配制的抗IFN $\gamma$单克隆抗体(mAb; 克隆AN18，$15\mathrm{g/mL}$)，$4^{\circ}C$孵育$16\mathrm{h}$。cRPMI封闭$(2\mathrm{h},37^{\circ}\mathrm{C})$后PBS洗涤。每孔加入$0.5\times10^{5}$肺细胞或$1\times10^{5}$脾细胞，并加入cRPMI配制的抗原(见具体实验)或$5\mathrm{g/mL}$刀豆蛋白A(Sigma; Melbourne, Australia)。$37^{\circ}C$、$5%$ $\mathrm{CO}_{2}$条件下孵育过夜。使用含$0.01%$ Tween-20的PBS洗涤后，加入PBS配制的抗IFN $\gamma$-生物素mAb(克隆XMG-1.2，$2.5~{\mathrm{g/mL}}$)，$37^{\circ}C$孵育$2\mathrm{h}$。洗涤后每孔加入1:1000 PBS稀释的亲和素-碱性磷酸酶(Sigma)，室温孵育$45\mathrm{min}$。洗涤后每孔加入NPP缓冲液稀释的$100~{\mathrm{L}}$ AP底物溶液(Bio-Rad, Sydney, Australia)，显色后水洗。使用AID ELISpot读数仪(Melbourne, Australia)配合ELISpot Reader 6.0软件计数斑点。
2.8. Statistical Analysis
2.8. 统计分析
Data were analysed using GraphPad version 7.00. Student’s $t.$ -test was used for comparing the statistical significance of differences between two sets of data and corrected for multiple comparisons using the Holm–Sidak method, and ANOVA was used to compare three or more groups. Significant differences were denoted as $^{}p<0.05,$ $^{\cdot\ast}p<0.01$ , $^{}p<0.001$ , $^{****}p<0.0001$ .
数据使用GraphPad 7.00版本进行分析。两组数据间的统计学差异显著性采用Student's $t.$ 检验，并通过Holm-Sidak方法进行多重比较校正；三组及以上数据比较采用ANOVA。显著性差异标记为 $^{}p<0.05$、$^{\cdot\ast}p<0.01$、$^{}p<0.001$ 和 $^{****}p<0.0001$。
3. Results
3. 结果
3.1. $C D4^T$ Cells Express CXCR3 in Response to Pulmonary Vaccination with rIAV
3.1. 肺部接种 rIAV 后 CD4+ T 细胞表达 CXCR3
To determine whether CXCR3 contributes to the cellular immune response to PR8.p25 vaccination, we immunized heterozygous CiBER mice, in which eGFP is expressed by $50%$ of CXCR3-expressing cells, with $\mathrm{i}/\mathrm{n}$ PR8.p25, and analysed CXCR3 expression on $\mathrm{CD}3^{+}$ $\mathrm{CD4^{+}}$ T cells (Figure 1A,B) and $\mathtt{p}25$ -specific ${\cal C}\mathrm{D}3^{+}{\cal C}\mathrm{D}4^{+}\mathrm{t}$ et+ T cells (Figure 1C,D) by flow cytometry. In the lungs, the frequency of T cells expressing CXCR3 peaked
为确定CXCR3是否参与PR8.p25疫苗接种的细胞免疫应答，我们用PR8.p25经鼻(i/n)免疫杂合CiBER小鼠(其中50%的CXCR3表达细胞会同时表达eGFP)，并通过流式细胞术分析CXCR3在CD3+CD4+ T细胞(图1A,B)和p25特异性CD3+CD4+ T细胞(图1C,D)上的表达。在肺部，表达CXCR3的CD3+CD4+ T细胞频率在...
7 days p.i. and declined until day 42, when $\mathrm{CXCR3^{+}C D4^{+}}$ T cells remain+ed m+ore frequent tehnatenr iend tuhnei lmumngusn iinz erde s(p0o dnasey st op .riI.)A Vm iPcRe 8(-Fpi2g5u (reF i1gEu)r. ${\mathrm{CD}}3^{+}{\mathrm{CD}}8^{+}$ hTo cweellds aa lssiom eilnatre rpeadtt etrhne lungs in response to rIAV PR8-p25 (Figure S3A) and showed a si+milar pattern of CXCR3 expression (Figure S3B). Vaccine-antigen-specific $\mathrm{CD4^{+}}$ T cells were identified with $\mathrm{IA}^{\flat}$ -p25 MHC class II tetramers t+agged+ with PE, which bind to $\mathtt{p}25$ -specific T cell receptors. These p25-tet+ $\mathrm{CD4^{+}}$ T c+ells w+ere first detected after 7 days and a similar proportion of p25-tet+ $\mathrm{CD4^{+}}$ T cells expressed CXCR3 from 7 d.p.i. until 42 d.p.i. (Figure 1F).
感染后7天(p.i.)，CXCR3+CD4+ T细胞频率开始下降，直至第42天时仍高于肺部未感染组(p<0.05)(图1E)。CD3+CD8+ T细胞在rIAV PR8-p25感染肺部也呈现相似分布模式(图S3A)，其CXCR3表达谱与CD4+ T细胞类似(图S3B)。通过PE标记的IA^b-p25 MHC II类四聚体(可结合p25特异性T细胞受体)鉴定疫苗抗原特异性CD4+ T细胞。这些p25四聚体+CD4+ T细胞在感染后7天首次检出，且从7 d.p.i.至42 d.p.i.期间持续有相近比例的细胞表达CXCR3(图1F)。
Figure 1. CXCR3-eGFP expression by pulmonary $\mathrm{CD4^{+}}$ T cells following PR8.p25 immunization. CiBER $^{+/-}$ mice $(\mathtt{n}=3{-}4)$ were immunized with 20 pfu PR8.p25 i.n. and pulmonary lymphocytes were isolated 3, 7, 21 and 42 days p.i. CXCR3-eGFP expression was measured by flow cytometry on lung $\mathrm{CD4^{+}}$ T cells (A,B) and p25-specific tet+ $\mathrm{CD4^{+}}$ T cells (D,E). Data are shown as the means $\pm$ SEM for the proportion of total $\mathrm{CD4^{+}}$ T cells (C) and $\mathtt{p}25$ -specific tet+ $\mathrm{CD4^{+}}$ T cells (F) expressing CXCR3-eGFP and are representative of two independent experiments. The significance of differences between the initial and later time points was determined by one-way ANOVA with multiple comparisons testing $(^{}p<0.05,^{***}p<0.001)$ .
图 1: PR8.p25免疫后肺组织$\mathrm{CD4^{+}}$T细胞的CXCR3-eGFP表达。CiBER$^{+/-}$小鼠$(\mathtt{n}=3{-}4)$经20 pfu PR8.p25鼻内免疫后，分别于感染后3、7、21和42天分离肺淋巴细胞。通过流式细胞术检测肺组织$\mathrm{CD4^{+}}$T细胞(A,B)和p25特异性tet+$\mathrm{CD4^{+}}$T细胞(D,E)的CXCR3-eGFP表达水平。数据显示为表达CXCR3-eGFP的总$\mathrm{CD4^{+}}$T细胞比例(C)及p25特异性tet+$\mathrm{CD4^{+}}$T细胞比例(F)的平均值$\pm$标准误，结果代表两次独立实验。采用单因素方差分析(ANOVA)进行多重比较检验，评估初始时间点与后续时间点的差异显著性$(^{}p<0.05,^{***}p<0.001)$。
We used intra vascular staining (IVS) and CD69 expression to identify tissue-resident $\mathrm{CD4^{+}}$ TRM $(\mathrm{CD3^{+}C D4^{+}C D69^{+}C D45I V^{-}})$ in the lung parenchyma 6 weeks after PR8- $\mathrm{p}25$ immunization (Figure 2A–F). A significantly higher proportion of p are nch y mal $\mathrm{CD4^{+}}$ TRM expressed CXCR3 in comparison to total lung $\mathrm{CD4^{+}}$ T cells (Figure 2G), and $50%$ of ${\mathfrak{p}}2{\mathfrak{H}}{-}{\mathrm{tet}}^{+}$ $\mathrm{CD4^{+}}$ TRM were eGFP-positive in the heterozygous CiBER mice, indicating that nearly all antigen-specific $\mathrm{CD4^{+}}$ TRM recruited to the lung expressed CXCR3 (Figure 2G).
我们采用血管内染色(IVS)和CD69表达来鉴定PR8-p25免疫6周后肺实质中的组织驻留CD4+ TRM(CD3+CD4+CD69+CD45IV−)(图2A-F)。与肺总CD4+ T细胞相比，肺实质CD4+ TRM中表达CXCR3的比例显著更高(图2G)，且在杂合CiBER小鼠中50%的p2H-tet+ CD4+ TRM呈eGFP阳性，表明几乎所有募集至肺部的抗原特异性CD4+ TRM都表达CXCR3(图2G)。
Figure 2. CXCR3-eGFP expression by $\mathrm{T}{\mathrm{RM}}$ after PR8.p25 immunization. $\mathrm{CiBER^{+/-}}$ mmiciec e( $(\mathrm{n}=3)$ wweerree immunized with 20 pfu PR8.p25 i.n. and pulmonary lymphocytes were isolated 42 days p.i. following intra vascular staining to identify lung-resident T cells. CXCR3-eGFP expression was examined on $\mathrm{CD44^{+}C D4^{+}}$ T cells (A,B), $\mathrm{CD69^{+}I V^{-}C D44^{+}C D4^{+}T_{R M}(C,D)},$ and $\mathtt{p}25$ -specific tet+ $\mathrm{CD4^{+}}$ T cell (E,F). CXCR3-eGFP expression on lung $\mathrm{CD4^{+}}$ T cells, $\mathrm{CD4^{+}}$ TRM and $\mathrm{CD4^{+}}$ tet+ $\mathrm{T}_{\mathrm{RM}}$ was then compared (G). Data are shown as the means $\pm$ SEM and are representative of two independent experiments. The significance of differences between the groups was determined by one-way ANOVA $(^{}p<0.05,$ $^{***}p<0.001.$ ).
图 2: PR8.p25免疫后组织驻留记忆T细胞(TRM)的CXCR3-eGFP表达。CiBER+/-小鼠(n=3)经鼻内接种20pfu PR8.p25后，于感染后42天分离肺淋巴细胞，并通过血管内染色鉴定肺组织驻留T细胞。检测了CXCR3-eGFP在CD44+CD4+T细胞(A,B)、CD69+IV-CD44+CD4+TRM(C,D)以及p25特异性tet+CD4+T细胞(E,F)上的表达。随后比较了CXCR3-eGFP在肺CD4+T细胞、CD4+TRM和CD4+tet+TRM中的表达水平(G)。数据以均值±标准误表示，结果来自两次独立实验。组间差异显著性通过单因素方差分析确定(*p<0.05, ***p<0.001)。
3.2. CXCR3 Is Not Required for the Recruitment of $C D4^T$ Cells to the Lungs Following rIAV Vaccination
3.2. CXCR3 不是 rIAV 疫苗接种后 $C D4^T$ 细胞募集至肺部所必需的
To investigate whether CXCR3 is necessary for the induction of a p25-specific $\mathrm{CD4^{+}}$ T cell response to PR8.p25 or the formation of pulmonary $\mathrm{CD4^{+}}$ $\mathrm{T_{RM}},$ we immunized $\mathrm{CXCR}3^{-7-}$ and WT mice $\mathrm{i}/\mathrm{n}$ With PR8.p25 and measured the $\mathrm{CD4^{+}}$ T cell response in the lungs, MLNs and spleens (Figure 3 and Figure S4A,C,E). At early time points, there were no significant differences in the total number of $\mathrm{CD4^{+}}$ T cells in the lungs or MLNs of $\mathrm{CXCR}3^{-/-}$ and WT mice, but a significant increase in the number of $\mathrm{CD}8^{+}$ T cells in the lungs and spleens of $\mathrm{CXCR}3^{-/-}$ mice compared to WT mice (Figure S4B,F). There was also a significant increase in the total number of $\mathrm{CD4^{+}}$ T cells in the spleens of $\mathrm{CXCR}3^{-/-}$ mmiiccee compared to WT mice (Figure S4E). We observed a trend towards greater numbers of $\mathrm{CD4^{+}}$ T cells in the lungs and MLNs of $\mathrm{CXCR}3^{-/-}$ mice at days 3 and 5 post-immunization, but this was abrogated at day 7 (Figure S4A,C). By day 11, we could detect vaccine-antigenspecific $\mathsf{p}25\mathrm{-tet^{+}~C D4^{+}}$ T cells. Equivalent numbers of these p25-specific cells were present in the lungs of $\mathrm{CXCR}3^{-/-}$ and WT mice at day 11 p.i. (Figure 3A). At later time points, however, there were significantly more p25-tet+ $\mathrm{CD4^{+}}$ T cells in the lungs, MLNs and spleens of $\mathrm{CXCR}3^{-/-}$ compared to WT mice (Figure 3A–C).
为探究CXCR3对PR8.p25诱导的p25特异性$\mathrm{CD4^{+}}$T细胞应答及肺$\mathrm{CD4^{+}}$$\mathrm{T_{RM}}$形成的必要性，我们通过$\mathrm{i}/\mathrm{n}$途径免疫$\mathrm{CXCR}3^{-/-}$与野生型(WT)小鼠，并检测肺、纵隔淋巴结(MLN)和脾脏中的$\mathrm{CD4^{+}}$T细胞应答(图3和图S4A,C,E)。早期时间点显示，$\mathrm{CXCR}3^{-/-}$与WT小鼠肺或MLN中$\mathrm{CD4^{+}}$T细胞总数无显著差异，但$\mathrm{CXCR}3^{-/-}$小鼠肺和脾脏中$\mathrm{CD}8^{+}$T细胞数量显著高于WT组(图S4B,F)。$\mathrm{CXCR}3^{-/-}$小鼠脾脏$\mathrm{CD4^{+}}$T细胞总数也显著增加(图S4E)。免疫后第3-5天观察到$\mathrm{CXCR}3^{-/-}$小鼠肺和MLN中$\mathrm{CD4^{+}}$T细胞数量增多趋势，但第7天时消失(图S4A,C)。至第11天可检测疫苗抗原特异性$\mathsf{p}25\mathrm{-tet^{+}~C D4^{+}}$T细胞，此时$\mathrm{CXCR}3^{-/-}$与WT小鼠肺中p25特异性细胞数量相当(图3A)。但在后期时间点，$\mathrm{CXCR}3^{-/-}$小鼠肺、MLN和脾脏中的p25-tet+$\mathrm{CD4^{+}}$T细胞数量显著高于WT组(图3A–C)。
Figure 3. Increased numbers of $\mathtt{p}25$ tetramer+ $\mathrm{CD4^{+}}$ T cells in $\mathrm{CXCR}3^{-/-}$ mice after PR8.p25 PiRm8.pm25u in.in.z aatnido cne.l $\mathrm{CXCR}3^{-/-}$ d afrnodm tWheTi r mluincges , $(\mathrm{n}=4{-}5)$ ) d wspelreee nism atm 11u, n2i7z aendd 3w8i th 20 pfu PR8.p25 i.n. and cells were isolated from their lungs, MLNs and spleens at 11, 27 and 38 days post-immunization and analysed by flow cytometry. p25-specific tet+ ${\cal C}{\mit\mathrm{D}}3^{+}{\cal C}{\mit\mathrm{D}}4^{+}$ T cells were measured in the lungs (A), MLNs (B) and spleens (C). Data are shown as the means $\pm$ SEM and are representative of two *i pn d< e0.p0e5,n *d* ep n<t 0e.0x1p, e**r*i pm <e 0n.t0s0. 1)T. he significance of differences between the groups at each time point was determined by Student’s $t$ -test $^{}p<0.05_{.}$ , $^{}p<0.01$ , $^{***}p<0.001.$ ).
图 3: CXCR3缺陷小鼠在PR8.p25感染后肺部、MLN和脾脏中p25四聚体+CD4+ T细胞数量增加。WT和CXCR3-/-小鼠(n=4-5)通过鼻内接种20 pfu PR8.p25，分别在免疫后11、27和38天分离其肺、MLN和脾脏细胞，通过流式细胞术分析。肺(A)、MLN(B)和脾脏(C)中p25特异性tet+CD3+CD4+ T细胞的检测结果。数据以均值±SEM表示，代表两次独立实验(*p<0.05, **p<0.01, ***p<0.001)。各时间点组间差异显著性通过Student t检验确定。
We then examined the impact of CXCR3 deficiency on retention of antigen-specific $\mathrm{CD69^{+}C D44^{+}T_{R M}}$ I in the lung parenchyma following PR8.p25 immunization. Gating analysis was used to identify $\mathrm{CD4^{+}}$ T cells (Figure S1) that were $\mathrm{CD45IV^{-}C D69^{+}}$ TRM (Figure 4A) and $\mathtt{p}25$ -specific (Figure 4B). Strikingly, there were significantly more p25-tet+ $\mathrm{CD4^{+}}$ TRM in the lungs of $\mathrm{CXCR}3^{-/-}$ than in WT mice (Figure $4C$ ). Therefore, although CXCR3 is expressed on lung memory T cells following pulmonary immunization, the development and retention of lung-resident $\mathrm{CD4^{+}}$ TRM is independent of CXCR3 expression.
我们随后研究了CXCR3缺陷对PR8.p25免疫后肺实质中抗原特异性$\mathrm{CD69^{+}C D44^{+}T_{R M}}$ I细胞滞留的影响。通过门控分析鉴定$\mathrm{CD4^{+}}$ T细胞（图S1），这些细胞为$\mathrm{CD45IV^{-}C D69^{+}}$ TRM（图4A）且具有$\mathtt{p}25$特异性（图4B）。值得注意的是，$\mathrm{CXCR}3^{-/-}$小鼠肺组织中p25四聚体阳性$\mathrm{CD4^{+}}$ TRM细胞数量显著多于野生型小鼠（图4C）。因此，尽管肺部免疫后记忆T细胞会表达CXCR3，但肺驻留$\mathrm{CD4^{+}}$ TRM细胞的发育和滞留并不依赖于CXCR3的表达。
To determine whether $\mathrm{CXCR}3^{-/-}$ mice had more severe PR8.p25 infections that contributed to their p25-specific $\mathrm{CD4^{+}}$ T cell responses, we monitored the weights of the infected $\mathrm{CXCR}3^{-/-}$ and WT mice. Both strains lost and regained similar amounts of weight, indicating their infections were equally severe (Figure S5). We also analysed viral loads in both strains by plaque assay at 3 and 7 days p.i and found no difference between their viral loads.
为确定$\mathrm{CXCR}3^{-/-}$小鼠是否因更严重的PR8.p25感染而增强其p25特异性$\mathrm{CD4^{+}}$T细胞反应，我们监测了感染小鼠的体重变化。两种基因型小鼠的体重减轻与恢复程度相似，表明感染严重程度相当（图S5）。此外，我们通过噬斑测定法在感染后第3天和第7天检测了病毒载量，发现两组间无显著差异。
Figure 4. Increased numbers of $\mathtt{p}25$ -specific tetramer+ $\mathrm{CD4^{+}\mathrm{T_{RM}}}$ CinX $\mathrm{CXCR}3^{-/-}$ mice after PR8.p25 immunization. ${\mathrm{CXCR}}3^{-/-}$ and WT mice $(\mathbf{n}=5)$ were immunized with 20 pfu PR8.p25 i.n. and cells were isolated from their lungs 38 days post-immunization and analysed by flow cytometry. Following $\mathtt{p}25$ gating on $\mathrm{CD4^{+}}$ T cells, $\mathrm{T}{\mathrm{RM}}$ and p25-specific specific tet+ ${\cal C}{\mit\mathrm{D}}3^{+}{\cal C}{\mit\mathrm{D}}4^{+}$ T cells were identified by IVS (A) and tetramer staining (B), respectively, and tet+ $\mathrm{T}_{\mathrm{RM}}$ were compared between groups (C). Data are shown as the means $\pm$ SEM and are representative of two independent experiments. The significance of differences between the groups was determined by Student’s $t\mathrm{.}$ -test $(^{**}p<0.01)$ .
图 4: CXCR3基因缺失小鼠在PR8.p25免疫后p25特异性四聚体+CD4+组织驻留记忆T细胞数量增加。CXCR3−/−和野生型小鼠(n=5)经鼻内接种20 pfu PR8.p25后，于免疫后第38天分离肺组织细胞进行流式分析。(A)通过体内刺激法(IVS)鉴定CD4+T细胞中p25特异性TRM细胞；(B)通过四聚体染色鉴定p25特异性tet+CD3+CD4+T细胞；(C)比较两组间tet+TRM细胞比例。数据以均值±标准误表示，结果来自两次独立实验。组间差异显著性采用Student t检验(**p<0.01)。
3.3. Pulmonary T Cell Cytokine Responses Are Independent of CXCR3 Following rIAV Vaccination
3.3. rIAV 疫苗接种后肺 T 细胞细胞因子反应与 CXCR3 无关
To determine whether CXCR3 deficiency impaired the functional capacity of antigenspecific $\mathrm{CD4^{+}}$ T cells following PR8.p25 immunization, we measured the production of $\mathrm{IFN}\gamma,$ , TNF and IL-2 following the ex vivo re stimulation of lung, MLN and spleen cells mfroonma $\mathrm{CXCR}3^{-/-}$ and WT mice. We observed a trend towards increased percentages of pulmonary cytokine-producing $\mathrm{CD4^{+}}$ Te rcee lslisg fnriofimca $\mathrm{CXCR}3^{-/-}$ mice, although only $\mathrm{IFN}\gamma^{+}$ paanred $\operatorname{IL}-2^{+}$ single-positive cell frequencies were significantly elevated in $\mathrm{CXCR3^{-7-}}$ asmeidc e r ceo qm u ep narc ie eds toof pWoTl yfmuicnec t(iFoingaul rCe D54A+) .T Tcheell sM aLnNd sa lal nIdF sNpγl-eseencsr eotif $\mathrm{CXCR}3^{-/-}$ elml iscue bsshetosw aesd winelclr easa sIeLd- 2f+r se iq nu gel en-cpieoss iotif vpe oClyDf4u+n Tc ticoelnlsa l( $\mathrm{CD4^{+}}$ e T5 cB,elCl)s. and all $\mathrm{IFN}\gamma$ -secreting $\mathrm{CD4^{+}}$ T cell subsets as well as $\operatorname{IL}-2^{+}$ single-positive $\mathrm{CD4^{+}}$ T cells (Figure 5B,C).
为确定CXCR3缺失是否影响PR8.p25免疫后抗原特异性$\mathrm{CD4^{+}}$T细胞的功能，我们检测了$\mathrm{CXCR}3^{-/-}$和野生型小鼠肺、纵隔淋巴结(MLN)及脾细胞离体再刺激后$\mathrm{IFN}\gamma$、TNF和IL-2的产生情况。尽管仅$\mathrm{IFN}\gamma^{+}$和$\operatorname{IL}-2^{+}$单阳性细胞频率在$\mathrm{CXCR3^{-7-}}$小鼠肺中显著升高（图5A），但我们观察到$\mathrm{CXCR}3^{-/-}$小鼠肺细胞因子产生型$\mathrm{CD4^{+}}$T细胞比例呈增加趋势。MLN和脾脏中所有$\mathrm{IFN}\gamma$分泌型$\mathrm{CD4^{+}}$T细胞亚群及$\operatorname{IL}-2^{+}$单阳性$\mathrm{CD4^{+}}$T细胞频率均显著上升（图5B,C）。
Figure 5. Cytokine expression in response to $\mathtt{p}25$ 8 .ipn2 5P-iRm8.mp2u5n-iizmedm uC n Xi Cz eRd 3− $\mathrm{CXCR}3^{-/-}$ mice. a $\mathrm{CXCR}3^{-/-}$ and WT mice $(\mathrm{n}=5)$ ) were immunized with 20 pfu PR8.p25 i.n. and cells were isolated from their lungs, MLNs and spleens 38 days post-immunization. They were stimulated with $\mathtt{p}25$ overnight and analysed by flow cytometry. Boolean gating analysis was used to detect $\mathrm{CD}3^{+}$ $\mathrm{CD4^{+}}$ T cells that expressed ${\mathrm{IFN}}{-}\gamma,$ TNF $\propto$ and IL-2. Single- and multi-cytokine producing $\mathrm{CD}3^{+}$ $\mathrm{CD4^{+}}$ T cells were measured in the lungs (A), MLNs (B) and spleens (C). Data are shown as the means $\pm$ SEM and are representative of two independent experiments. The significance of differences between the groups was determined by Student’s $t\mathrm{.}$ -test $(^{\ast}p<0.05,^{\ast\ast}p<0.01)$ 1.6
图 5: 响应 $\mathtt{p}25$ 的细胞因子表达。$\mathrm{CXCR}3^{-/-}$ 和野生型 (WT) 小鼠 $(\mathrm{n}=5)$ 经鼻内免疫 20 pfu PR8.p25 后，在免疫后第 38 天分离其肺、纵隔淋巴结 (MLN) 和脾脏细胞。这些细胞经 $\mathtt{p}25$ 过夜刺激后，通过流式细胞术进行分析。采用布尔门控分析检测表达 ${\mathrm{IFN}}{-}\gamma$、TNF $\propto$ 和 IL-2 的 $\mathrm{CD}3^{+}$ $\mathrm{CD4^{+}}$ T 细胞。在肺 (A)、纵隔淋巴结 (B) 和脾脏 (C) 中测量了单细胞因子和多细胞因子产生的 $\mathrm{CD}3^{+}$ $\mathrm{CD4^{+}}$ T 细胞。数据以均值 $\pm$ 标准误表示，并代表两次独立实验。组间差异显著性通过 Student's $t\mathrm{.}$ 检验确定 $(^{\ast}p<0.05,^{\ast\ast}p<0.01)$。
Additionally, we performed ELISpot analysis to compare T cell $\mathrm{IFN}\gamma$ responses to the $\mathrm{NP}_{366-374}$ and $\mathtt{p}25$ epitopes of the PR8.p25 vaccine, which are recognised by $\mathrm{CD8^{+}}$ and $\mathrm{CD4^{+}}$ T c+ell3s66,– 3 r 74 esp ect iv ely. There were no differences between anti-NP and anti $\mathrm{p}25\mathrm{IFN}\gamma$ secreting responses in the lungs (Figure 6A) or anti $\mathrm{p}25\mathrm{IFN}\gamma$ responses in the spleens (Figure 6B). Significantly more sp leno cyte s produced $\mathrm{IFN-}\gamma$ in response to NP in $\mathrm{CX\bar{C}R3^{-/-}}$ mice than in WT mice (Figure 6B). These results demonstrate that the absence of CXCR3 does not impair T cell cytokine production.
此外，我们通过ELISpot分析比较了T细胞对PR8.p25疫苗中$\mathrm{NP}_{366-374}$和$\mathtt{p}25$表位的$\mathrm{IFN}\gamma$反应，这两个表位分别由$\mathrm{CD8^{+}}$和$\mathrm{CD4^{+}}$T细胞识别。肺部抗NP与抗$\mathrm{p}25\mathrm{IFN}\gamma$分泌反应（图6A）或脾脏抗$\mathrm{p}25\mathrm{IFN}\gamma$反应（图6B）均无显著差异。与野生型小鼠相比，$\mathrm{CX\bar{C}R3^{-/-}}$小鼠脾细胞对NP产生$\mathrm{IFN-}\gamma$的反应显著增强（图6B）。这些结果表明，CXCR3的缺失不会损害T细胞因子的产生。
Figure 6. Lymphocytes from vaccinated $\mathrm{CXCR}3^{-/-}$ mice produce $\operatorname{IFN}-\gamma$ di n response to $\mathrm{CD4^{+}}$ CaDn8+d $\mathrm{CD}8^{+}$ T cell vaccine antigens. ELISpot analysis of lung (A) and spleen (B) cells isolated from $\mathrm{CXCR}3^{-/-}$ Ta nmdic e W(n T= 4m) i3c8 ed $(\mathrm{n}=4)$ 38 days p.i. with PR8.p25. Cells were stimulated with either no antigen, $\mathrm{H}{-}2\mathrm{D}^{\mathrm{b}}$ -restricted $\mathrm{NP}_{366-374}$ peptide recognised by $\mathrm{CD8^{+}}$ T cells, $\mathrm{I}{-}\mathrm{A}^{\mathrm{b}}$ -restricted $\mathtt{p}25$ recognized by $\mathrm{CD4^{+}}$ T cells, or con ca naval in A. Data are shown as the means $\pm$ SEM and are representative of two independent experiments. The significance of differences between the groups was determined by Student's $t$ -test $(^{**}p<0.01)$ .
图 6: 接种疫苗的 CXCR3-/- 小鼠淋巴细胞对 CD4+ 和 CD8+ T 细胞疫苗抗原产生 IFN-γ 应答。(A) 肺脏和 (B) 脾脏细胞 ELISpot 分析结果，样本来自 PR8.p25 感染 38 天后的 CXCR3-/- 小鼠 (n=4)。细胞分别采用以下刺激条件：无抗原、CD8+ T 细胞识别的 H-2Db 限制性 NP366-374 肽段、CD4+ T 细胞识别的 I-Ab 限制性 p25 肽段或刀豆蛋白 A。数据以均值 ± 标准误表示，结果来自两次独立实验。组间差异显著性通过 Student t 检验确定 (**p<0.01)。
3.4. CXCR3 Provides a Competitive Adoantage to $C D4^{+}$ T Cell Responses to rIAV Vaccination To determine whether CXCR3 confers a competitive advantage in the $\mathrm{CD4^{+}}$ T cell response to PR8.p25, we adoptively transferred 25,000 WT P25 cells and $25,000\mathrm{CXCR3^{-/-}}$
3.4. CXCR3为$CD4^{+}$T细胞对rIAV疫苗接种的应答提供竞争优势
为确定CXCR3是否在$\mathrm{CD4^{+}}$T细胞对PR8.p25的应答中赋予竞争优势，我们过继性转移了25,000个野生型P25细胞和25,000个$\mathrm{CXCR3^{-/-}}$细胞。
P25 cells into WT mice, immunized the mice with $\mathrm{PR}8.\mathrm{p}25\mathrm{i}/\mathrm{n}$ and measured the recruitment of antigen-specific $\mathrm{T}$ cells to the lungs via flow cytometry (Figure 7A,B). Both $\mathrm{CXCR}3^{-/-}$ and WT antigen-specific $\mathrm{T}$ cells were recruited to the lungs in similar numbers after 7 days (Figure 7C). From day 11 onwards, however, a smaller proportion of $\mathrm{CXCR}3^{-/-}$ than WT antigen-specific cells remained in the lungs, and these differences were significant at day 11 and day 42 (Figure 7C). There were no significant differences between $\mathrm{CXCR}3^{-/-}$ and WT antigen-specific T cells present at any time points in the MLNs or spleens (Figure 7D,E). Furthermore, there were significantly fewer $\mathtt{p}25$ antigen-specific $\mathrm{CX(CR3^{-/-}C D45I V^{-}C D69^{+}C D3^{+}C D4^{+}T_{R M}}$ in the lungs compared with WT P25 T cells at day 42 (Figure 7F). Therefore, although CXCR3 was not required for retention of antigenspecific $\mathrm{T}_{\mathrm{RM}}$ in the lungs, the presence of this receptor provided a competitive advantage for the recruitment $\mathrm{CD4^{+}}$ T cells into the lung and their retention as antigen-specific TRM.
将P25细胞注入野生型(WT)小鼠体内，用$\mathrm{PR}8.\mathrm{p}25\mathrm{i}/\mathrm{n}$免疫小鼠后，通过流式细胞术检测抗原特异性$\mathrm{T}$细胞向肺部的募集情况(图7A,B)。第7天时，$\mathrm{CXCR}3^{-/-}$和WT抗原特异性$\mathrm{T}$细胞在肺部的募集数量相当(图7C)。但从第11天起，$\mathrm{CXCR}3^{-/-}$抗原特异性细胞在肺部的驻留比例显著低于WT组，这种差异在第11天和第42天具有统计学意义(图7C)。在纵隔淋巴结(MLNs)和脾脏中，$\mathrm{CXCR}3^{-/-}$与WT抗原特异性T细胞的数量在所有时间点均无显著差异(图7D,E)。此外，第42天时肺组织中$\mathtt{p}25$抗原特异性$\mathrm{CX(CR3^{-/-}C D45I V^{-}C D69^{+}C D3^{+}C D4^{+}T_{R M}}$细胞数量显著少于WT P25 T细胞(图7F)。因此，虽然CXCR3并非肺部驻留抗原特异性$\mathrm{T}_{\mathrm{RM}}$细胞的必需条件，但该受体的存在能为$\mathrm{CD4^{+}}$T细胞向肺部的募集及后续转化为抗原特异性TRM提供竞争优势。
FF i igg u ur ree 77. . ${\mathrm{CXCR}}3^{-/-}$ $\mathtt{p}25$ -specific $\mathrm{CD4^{+}}$ T cells have a competitive disadvantage compared to WT $\mathtt{p}25$ -specific $\mathrm{CD4^{+}}$ T cells for recruitment to and retention in the lung. $25,000\mathrm{CXCR3^{-/-}}$ and WT $\mathrm{P}25\mathrm{CD}4^{+}$ T cells were transferred to C57Bl/6 mice $(\mathrm{n}=4{-}6)$ ) $24\mathrm{h}$ prior to i.n. immunization with 20 pfu of PR8.p25 (A). After 7, 10, 21 and 42 days, lungs, MLNs and spleens were harvested, and cells were stained and analysed by flow cytometry. The congenic markers $\mathrm{CD45.1}$ and CD45.2 were used to identify WT and CXCR3KO P25 cells (B). WT and CXCR3KO P25 cells were quantified in the (lFu).n gDsa (taC )a, reM sLhNos w(nD )a sa nthd es pml eee an nss (±E )S. EWMT aanndd aCreX Cr eRp 3 rKe sOe nPt2a5t $\mathrm{T}_{\mathrm{RM}}$ f wtewroe iq nu dae np tie fined de innt tehxep leurinmgse natt sd. ay 42. (F). Data are shown as the means $\pm$ SEM and are representative of two independent experiments. The significance of differences between the groups was determined by Student's $t\cdot$ -test $(^{*}p<0.05,$ $^{**}p<0.01$ ).
图 7: CXCR3缺失的p25特异性CD4+ T细胞在招募和驻留肺部方面相比野生型p25特异性CD4+ T细胞具有竞争劣势。(A)在鼻内免疫PR8.p25病毒(20 pfu)前24小时，将25,000个CXCR3敲除和野生型P25 CD4+ T细胞过继转移至C57Bl/6小鼠(n=4-6)。(B)流式细胞术采用CD45.1和CD45.2同源标志物区分野生型与CXCR3敲除的P25细胞。(C-E)分别量化肺脏(C)、纵隔淋巴结(D)和脾脏(E)中的WT与CXCR3KO P25细胞数量。(F)第42天时肺部驻留记忆T细胞(TRM)的表型鉴定。数据以均值±标准误表示，结果来自两次独立实验。组间差异显著性采用Student t检验评估(*p<0.05，**p<0.01)。
4. Discussion
4. 讨论
tVya bcceicnaeu-sien dofu tcheedi $\mathrm{T}{\mathrm{RM}}$ i dh arevsep roencseen ttloy Mb.e teunb e prc ru o lpo so is s ecdh aalsl ean gceo rarte tlahtee siotfe porf o it ne fc etc itv ioe nT B immunity because of their rapid response to M. tuberculosis challenge at the site of infection and their role in long-term protection [9,21,39]. In human TB patients, $\mathrm{T}{\mathrm{RM}}$ accumulate at all infection sites, are highly activated, and elicit a rapid multi functional cytokine response to re stimulation with M. tuberculosis in vitro [40]. While the expression of CD69 and CD103 is generally accepted as identifying TRM, the need for additional well-validated and accurate TRM markers [41,42] has prompted the evaluation of $\mathrm{CD49a}$ , CXCR6, CXCR3, CD101, PD-1, CD62L, KLRG1, and CX3CR1 in various studies [43]. This study focuses on CXCR3, which is highly expressed by $\mathrm{T}_{\mathrm{RM}}$ and has been considered necessary for their development and retention [30,44,45]+. The key findings of our study reveal that while the majority of vaccine-induced $\mathrm{CD4^{+}}$ TRM in the lungs does express CXCR3 in response to pulmona+ry immunization, CXCR3 expression is not necessary for the formation and retention of $\mathrm{CD4^{+}}$ TRM in the lungs.
tVya bcceicnaeu-sien dofu tcheedi $\mathrm{T}{\mathrm{RM}}$ i dh arevsep roencseen ttloy Mb.e teunb e prc ru o lpo so is s ecdh aalsl ean gceo rarte tlahtee siotfe porf o it ne fc etc itv ioe nT B immunity because of their rapid response to M. tuberculosis challenge at the site of infection and their role in long-term protection [9,21,39]. In human TB patients, $\mathrm{T}{\mathrm{RM}}$ accumulate at all infection sites, are highly activated, and elicit a rapid multi functional cytokine response to re stimulation with M. tuberculosis in vitro [40]. While the expression of CD69 and CD103 is generally accepted as identifying TRM, the need for additional well-validated and accurate TRM markers [41,42] has prompted the evaluation of $\mathrm{CD49a}$ , CXCR6, CXCR3, CD101, PD-1, CD62L, KLRG1, and CX3CR1 in various studies [43]. This study focuses on CXCR3, which is highly expressed by $\mathrm{T}_{\mathrm{RM}}$ and has been considered necessary for their development and retention [30,44,45]+. The key findings of our study reveal that while the majority of vaccine-induced $\mathrm{CD4^{+}}$ TRM in the lungs does express CXCR3 in response to pulmona+ry immunization, CXCR3 expression is not necessary for the formation and retention of $\mathrm{CD4^{+}}$ TRM in the lungs.
CXCR3 plays a major role in immune responses to lung pathogens, being rapidly up regulated after T cell activation [46] and facilitating the migration of T cells to the lungs and draining lymph nodes [47]. CXCR3 ligands CXCL9, CXCL10 and CXCL11 are highly expressed in the lung in response to influenza [48] as well as M. tuberculosis [29,49] infection. Serum levels of CXCR3 ligands, in particular CXCL10 (or IP-10), have been studied as potential biomarkers of active TB disease [50] and the rapid response to TB treatment [51]. CXCR3 may also play a role in the induction of T cell memory as antigen recall responses from $\mathrm{CD4^{+}}$ and $\mathrm{CD8^{+}}$ TCM and $\mathrm{T_{EM}}$ were largely restricted to ${\mathrm{CXCR}}3^{+}$ cell subsets [52,53]. In influenza infection, CXCR3 is important for promoting T follicular helper $(\mathrm{T_{FH}})$ cell activity and antibody production [48], and in mouse TB models it is a marker for a protective antigen-specific $\mathrm{CD4^{+}}$ T cell subset [31]. In the non-human primate model of latent TB infection, there were high frequencies of $\mathrm{CXCR3^{+}C D4^{+}}$ T cells in lung granulomas, inversely proportional to the $M.$ . tuberculosis bacterial load, and CXCR3 was co-expressed on the antigen-specific T cells producing $\mathrm{IFN}\gamma$ and IL-17 [54]. Paradoxically, the disruption of CXCR3-CXCL11 signalling in zebrafish increases resistance to mycobacteria l infection [25], and $\mathrm{CXCR}3^{-/-}$ mice with a BALB/c background exhibited improved control of chronic M. tuberculosis infection [55]. This suggests that while CXCR3 expression may appear to correlate with protective immunity to TB infection, this relationship is not causal.
CXCR3在肺部病原体免疫应答中起主要作用，T细胞激活后会迅速上调[46]，并促进T细胞向肺部和引流淋巴结迁移[47]。CXCR3配体CXCL9、CXCL10和CXCL11在流感[48]及结核分枝杆菌[29,49]感染时于肺部高表达。血清CXCR3配体（尤其是CXCL10/IP-10）水平已被研究作为活动性结核病[50]及结核治疗快速响应[51]的潜在生物标志物。CXCR3可能还参与T细胞记忆诱导，因为来自$\mathrm{CD4^{+}}$和$\mathrm{CD8^{+}}$中央记忆T细胞（TCM）与效应记忆T细胞（$\mathrm{T_{EM}}$）的抗原回忆应答主要局限于${\mathrm{CXCR}}3^{+}$细胞亚群[52,53]。流感感染中，CXCR3对促进滤泡辅助性T细胞（$\mathrm{T_{FH}}$）活性和抗体产生至关重要[48]；在小鼠结核模型中，它是保护性抗原特异性$\mathrm{CD4^{+}}$T细胞亚群的标志物[31]。在非人灵长类潜伏结核感染模型中，肺肉芽肿内$\mathrm{CXCR3^{+}C D4^{+}}$T细胞频率与结核分枝杆菌载量呈负相关，且CXCR3在产生$\mathrm{IFN}\gamma$和IL-17的抗原特异性T细胞上共表达[54]。矛盾的是，斑马鱼中CXCR3-CXCL11信号阻断会增强对分枝杆菌感染的抵抗力[25]，而BALB/c背景的$\mathrm{CXCR}3^{-/-}$小鼠对慢性结核分枝杆菌感染的控制能力更强[55]。这表明虽然CXCR3表达看似与结核感染保护性免疫相关，但二者并非因果关系。
In this study CXCR3 was expressed by $\mathrm{CD4^{+}}$ T cells in the lungs following rIAV immunization with PR8.p25 (Figure 1E), and maintained on rIAV-induced, p25-specific $\mathrm{CD4^{+}}$ T cells for at least six weeks post-immunization (Figure 1F). This is consistent with previous findings showing that mucosal, but not peripheral, TB vaccination results in the generation of antigen-specific $\mathrm{CXCR3^{+}C D4^{+}}$ T cells in the lung parenchyma [56]. The mice used in the current study were heterozygous CiBER females expressing the CXCR3 gene on both X chromosomes, but with eGFP under the control of the CXCR3 promoter on one chromosome. Because the Lyon effect results in gene expression in individual cells from only one X chromosome at random [57], only half of the T cells in the CiBEReGFP/WT mice express the eGFP reporter gene when CXCR3 is expressed. Unlike surface staining analysis, which only detects CXCR3 molecules present on the cell surface, this model allows for the detection of all cells translating CXCR3-coding mRNA. Approximately $40%$ of $\mathtt{p}25$ -tetramer+ $\mathrm{CD4^{+}}$ T cells maintained eGFP expression until 42 d.p.i (Figure 1F), indicating that $80%$ of $\mathtt{p}25$ -specific cells expressed CXCR3 for at least six weeks following vaccination. As CXCR3 is believed to be required for T cell migration into the lung [30] and CXCR3 deficiency has been reported to decrease the rate of $\mathrm{CD4^{+}}$ T cell entry to the lung parenchyma by from five- to ten-fold [27], we examined the impact of deleting CXCR3 expression in this model of mucosal immunization.
本研究中，PR8.p25接种后肺部CD4+ T细胞表达CXCR3（图1E），且这种表达在rIAV诱导的p25特异性CD4+ T细胞上持续至少六周（图1F）。这与先前研究结果一致，即黏膜（而非外周）结核疫苗接种会在肺实质中产生抗原特异性CXCR3+CD4+ T细胞[56]。本研究使用的CiBER雌性杂合小鼠在两个X染色体上均携带CXCR3基因，但其中一个染色体的CXCR3启动子控制eGFP表达。由于Lyon效应导致单个细胞随机选择一条X染色体进行基因表达[57]，当CXCR3表达时，CiBEReGFP/WT小鼠中仅半数T细胞表达eGFP报告基因。与仅检测细胞表面CXCR3分子的表面染色分析不同，该模型可检测所有翻译CXCR3编码mRNA的细胞。约40%的p25四聚体+CD4+ T细胞持续表达eGFP至接种后42天（图1F），表明80%的p25特异性细胞在接种后至少六周内持续表达CXCR3。鉴于CXCR3被认为是T细胞迁移至肺部的必要条件[30]，且据报道CXCR3缺陷会使CD4+ T细胞进入肺实质的速率降低5-10倍[27]，我们在此黏膜免疫模型中研究了删除CXCR3表达的影响。
Unexpectedly, $\mathrm{CXCR}3^{-/-}$ mice had equivalent numbers of p25-specific $\mathrm{CD4^{+}}$ T cells to WT mice in the lungs 11 days after PR8.p25 immunization, and increased numbers of p25-specific $\mathrm{CD4^{+}}$ T cells at days 28 and 38 p.i. (Figure 3A). CXCR3 is up regulated upon activation of naïve T cells and remains highly expressed on Th1 $\mathrm{CD4^{+}}$ T cells [58,59]. In addition, interactions between CXCR3 and its ligands facilitate the movement of $\mathrm{CD4^{+}}$ Th1 cells to sites of inflammation [60]. However, our results indicate that the deletion of CXCR3 resulted in enhanced antigen-specific $\mathrm{CD4^{+}}$ T cell responses to rIAV immunization in the lungs, suggesting that the presence of CXCR3 signalling is not wholly beneficial in the context of mucosal TB vaccination. This is not true for vaccine-induced antigen-specific $\mathrm{CD}8^{+}$ T cells during M. tuberculosis infection in mice, which exhibit impaired entry to the lung parenchyma when treated with a CXCR3-blocking antibody [30]. Notably, we showed that CXCR3 expression was not necessary for the formation of $\mathrm{CD4^{+}}$ TRM, and the $\mathrm{CXCR}3^{-/-}$ mice generated more p25-specific $\mathrm{CD4^{+}}$ TRM than WT mice (Figure 4C). $\mathrm{CXCR}3^{-/-}$ mice also had more $\mathtt{p}25$ -specific $\mathrm{CD4^{+}}$ T cells in their spleens than WT mice at all studied time points (Figure 3C), indicating they had a larger systemic memory $\mathrm{CD4^{+}}$ T cell response in addition to their increased number of $\mathrm{T}{\mathrm{RM}}$ . One possible explanation for these differences is that, in $\mathrm{CXCR}3^{-/-}$ mice, $\mathrm{CD4^{+}}$ T cells employ compensatory mechanisms for lung-homing that enhance memory cell formation through altered tissue-homing or enhanced proliferation or survival. This has implications for future vaccine strategies, as techniques to suppress CXCR3 signalling using drugs or vaccines that prevent CXCR3 ligand expression may be employed with future vaccines to induce a larger systemic or resident memory $\mathrm{CD4^{+}}$ T cell response. This approach proved effective for $\mathrm{CD8^{+}}$ T cell responses in mice [61], where the temporary pharmaceutical inhibition of CXCR3 and CCR5 resulted in enhanced $\mathrm{CD}8^{+}$ T cell responses. When $\mathrm{CXCR}3^{-/-}$ p25-specific $\mathrm{CD4^{+}T}$ cells were adoptively transferred into WT hosts along with WT p25-specific T cells, however, the CXCR3-deficient $\mathrm{CD4^{+}}$ T cells entered the lungs in reduced numbers compared to their WT counterparts at early and later time points p.i. (Figure 7C), and there was a marked reduction in the number of antigen-specific TRM in the lungs (Figure 7F). Furthermore, $\mathrm{CXCR}3^{-/-}$ and WT P25 cells remained in equal numbers in the MLNs and spleens at all time points (Figure 7D,E), in contrast with $\mathrm{CXCR}3^{-/-}$ mice, which had increased numbers of splenic $\mathtt{p}25.$ -specific $\mathrm{CD4^{+}}$ T cells (Figure 3C). This shows that $\mathrm{CXCR}3^{-/-}$ $\mathrm{CD4^{+}}$ T cells compete equally with their WT counterparts in differentiating to circulating memory phenotypes, but not in lung $\mathrm{T}_{\mathrm{RM}}$ formation. This may be because WT $\mathrm{CD4^{+}}$ T cells outcompete $\dot{\mathrm{{CXCR3^{-/-}}}}$ T cells in accessing cytokines or physical spaces needed for differentiation into $\mathrm{CD4^{+}}$ TRM. This contrasts with the study by Dhume et al. [62], who adoptively transferred T-bet-deficient antigen-specific $\mathrm{CD4^{+}}$ T cells with decreased CXCR3 expression, along with WT cells into mice infected with influenza A virus. They showed that T-bet-deficient cells entered the lungs in reduced numbers, similarly to the $\dot{\mathrm{CXCR3^{-/-}}}$ cells examined here. However, they first saw defective lung entry after 7 days, when we observed no differences. They also noted enhanced splenic memory cell formation by $\mathrm{T}\mathrm{-bet^{-/-}C D4^{+}}$ T cells, which did not occur with adoptively transferred $\mathrm{CXCR3^{-/-}C D4^{+}}$ T cells. These results expand upon observations that ${\mathrm{CXCR}}3^{+}{\mathrm{CD}}4^{+}$ T cells purified from the lungs of $M$ . tuberculosis-challenged mice will migrate back into the lungs [63,64], but the clinical impact of this competitive advantage of WT over $\mathrm{CXCR}3^{-/-}$ p25-specific $\mathrm{CD4^{+}}$ T cells remains to be determined.
出乎意料的是，在PR8.p25免疫后第11天，$\mathrm{CXCR}3^{-/-}$小鼠肺中p25特异性$\mathrm{CD4^{+}}$T细胞数量与野生型(WT)小鼠相当，而在感染后第28天和38天则出现增加(图3A)。CXCR3在初始T细胞激活时上调，并在Th1型$\mathrm{CD4^{+}}$T细胞中持续高表达[58,59]。此外，CXCR3与其配体的相互作用促进$\mathrm{CD4^{+}}$Th1细胞向炎症部位迁移[60]。但我们的结果表明，CXCR3缺失增强了肺部对rIAV免疫的抗原特异性$\mathrm{CD4^{+}}$T细胞应答，提示CXCR3信号在黏膜结核疫苗接种中并非完全有益。这与小鼠结核分枝杆菌感染中疫苗诱导的抗原特异性$\mathrm{CD}8^{+}$T细胞不同——使用CXCR3阻断抗体会削弱其进入肺实质的能力[30]。值得注意的是，我们发现CXCR3表达并非$\mathrm{CD4^{+}}$TRM形成的必要条件，且$\mathrm{CXCR}3^{-/-}$小鼠比WT小鼠产生更多p25特异性$\mathrm{CD4^{+}}$TRM(图4C)。在所有观察时间点，$\mathrm{CXCR}3^{-/-}$小鼠脾脏中的$\mathtt{p}25$特异性$\mathrm{CD4^{+}}$T细胞也多于WT小鼠(图3C)，表明除增加的$\mathrm{T}{\mathrm{RM}}$数量外，其全身性记忆$\mathrm{CD4^{+}}$T细胞应答也更显著。这些差异的可能解释是：$\mathrm{CXCR}3^{-/-}$小鼠的$\mathrm{CD4^{+}}$T细胞通过改变组织归巢或增强增殖/存活等代偿机制促进记忆细胞形成。这对未来疫苗策略具有启示意义——通过药物或疫苗抑制CXCR3信号或阻断其配体表达的技术，可能用于诱导更强的全身性或定居记忆$\mathrm{CD4^{+}}$T细胞应答。该方法在小鼠$\mathrm{CD8^{+}}$T细胞应答中已证实有效[61]，短暂抑制CXCR3和CCR5可增强$\mathrm{CD}8^{+}$T细胞应答。然而，当将$\mathrm{CXCR}3^{-/-}$p25特异性$\mathrm{CD4^{+}T}$细胞与WT p25特异性T细胞共同过继转移至WT宿主时，CXCR3缺陷的$\mathrm{CD4^{+}}$T细胞在感染早期和后期进入肺部的数量均少于WT细胞(图7C)，且肺中抗原特异性TRM数量显著减少(图7F)。此外，$\mathrm{CXCR}3^{-/-}$与WT P25细胞在MLN和脾脏中的数量始终相当(图7D,E)，这与$\mathrm{CXCR}3^{-/-}$小鼠脾脏$\mathtt{p}25.$特异性$\mathrm{CD4^{+}}$T细胞增加的现象(图3C)形成对比。这表明$\mathrm{CXCR}3^{-/-}$$\mathrm{CD4^{+}}$T细胞在分化为循环记忆表型时与WT细胞竞争力相当，但在肺$\mathrm{T}_{\mathrm{RM}}$形成中处于劣势，可能是WT$\mathrm{CD4^{+}}$T细胞在获取分化所需细胞因子或物理空间方面更具优势。这与Dhume等[62]的研究不同：他们过继转移了CXCR3表达降低的T-bet缺陷抗原特异性$\mathrm{CD4^{+}}$T细胞与WT细胞至甲型流感病毒感染小鼠，发现T-bet缺陷细胞肺部归巢减少（与本研究的$\dot{\mathrm{CXCR3^{-/-}}}$细胞类似），但肺部归巢缺陷在7天后才出现（而我们未观察到早期差异）。他们还发现$\mathrm{T}\mathrm{-bet^{-/-}C D4^{+}}$T细胞会增强脾脏记忆细胞形成，而过继转移的$\mathrm{CXCR3^{-/-}C D4^{+}}$T细胞无此现象。这些结果拓展了先前发现：从结核分枝杆菌感染小鼠肺部分离的${\mathrm{CXCR}}3^{+}{\mathrm{CD}}4^{+}$T细胞具有肺部再归巢能力[63,64]，但WT细胞相对$\mathrm{CXCR}3^{-/-}$p25特异性$\mathrm{CD4^{+}}$T细胞的竞争优势对临床的影响仍需进一步研究。
CXCR3 plays a role in T cell migration but does not affect antigen recall responses in T cells. Lymphocytes from both $\mathrm{CXCR}\check{3}^{-/-}$ and WT mice vaccinated with PR8.p25 expressed $\mathrm{IFN-}\gamma$ in response to $\mathrm{CD4^{+}}$ and $\mathrm{CD8^{+}}$ T cell vaccine epitopes after 6 weeks (Figure 5). In addition, $\mathrm{CXC}\dot{\mathrm{R}}3^{-/-}$ mice displayed increased poly functional cytokine $\mathrm{CD4^{+}}$ T cell responses following $\mathtt{p}25$ stimulation compared to WT mice. Such poly functional T cell responses are associated with protection against $M$ . tuberculosis infection in BCG-vaccinated mice [65]; however, they do not correlate with protective immunity against TB in humans [7,66]. Furthermore, this study demonstrates that the number and functionality of antigen-specific $\mathrm{CD4^{+}}$ T cells in the lungs and the retention of antigen-specific $\mathrm{T}_{\mathrm{RM}}$ following mucosal TB immunization is independent of the chemokine receptor, CXCR3. In fact, memory $\mathrm{CD4^{+}}$ T cell responses to the pulmonary vaccine were increased in the absence of CXCR3. This finding is consistent with the observation that CXCR3-deficient BALB/c mice infected by aerosol with M. tuberculosis developed increased memory T cell responses that were associated with increased clearance of the mycobacteria at 12 and 24 weeks [55]. The mechanism by which CXCR3 regulates memory $\mathrm{CD4^{+}}$ T cell formation in response to rIAV vaccination or M. tuberculosis infection is unknown. CXCR3 expression did not affect $\mathrm{CD4^{+}}$ T cell entry into the lungs up to 11 days after immunization, but $\mathrm{CXCR}3^{-/-}$ mice developed increased p25-specific splenic $\mathrm{CD4^{+}}$ T cells at days 11 and 28, indicating that these underwent increased expansion or decreased contraction prior to this. This could be related to impaired T cell-DC interactions in lymph nodes, as has been reported previously [32]. To elucidate this mechanism, future studies could employ imaging techniques to assess $\mathrm{CD4^{+}}$ T cell local is ation with antigen in both the lymph nodes and lung, as well as the impact of CXCR3 deletion on early T cell expansion and differentiation.
CXCR3在T细胞迁移中发挥作用，但不影响T细胞的抗原回忆反应。接种PR8.p25疫苗的$\mathrm{CXCR}\check{3}^{-/-}$和野生型小鼠淋巴细胞在6周后均能针对$\mathrm{CD4^{+}}$和$\mathrm{CD8^{+}}$T细胞疫苗表位产生$\mathrm{IFN-}\gamma$应答(图5)。此外，与野生型小鼠相比，$\mathrm{CXC}\dot{\mathrm{R}}3^{-/-}$小鼠在$\mathtt{p}25$刺激后表现出更强的多功能细胞因子$\mathrm{CD4^{+}}$T细胞反应。这类多功能T细胞反应与BCG疫苗接种小鼠对$M$. tuberculosis感染的防护相关[65]，但与人体的结核病防护免疫无相关性[7,66]。本研究进一步表明，黏膜结核免疫后肺中抗原特异性$\mathrm{CD4^{+}}$T细胞的数量和功能，以及抗原特异性$\mathrm{T}_{\mathrm{RM}}$的存留均不依赖趋化因子受体CXCR3。事实上，缺乏CXCR3时对肺部疫苗的记忆$\mathrm{CD4^{+}}$T细胞反应反而增强。这一发现与先前观察结果一致：经气溶胶感染结核分枝杆菌的CXCR3缺陷BALB/c小鼠会增强记忆T细胞反应，并在12周和24周时加速分枝杆菌的清除[55]。CXCR3调控记忆$\mathrm{CD4^{+}}$T细胞形成以应对rIAV疫苗接种或结核分枝杆菌感染的机制尚不明确。免疫后11天内CXCR3表达不影响$\mathrm{CD4^{+}}$T细胞进入肺部，但$\mathrm{CXCR}3^{-/-}$小鼠在第11天和28天时脾脏p25特异性$\mathrm{CD4^{+}}$T细胞增多，表明此前经历了更强的扩增或更弱的收缩。这可能与淋巴结中T细胞-DC相互作用受损有关，如先前报道[32]。为阐明该机制，未来研究可采用成像技术评估淋巴结和肺中$\mathrm{CD4^{+}}$T细胞与抗原的共定位，以及CXCR3缺失对早期T细胞扩增和分化的影响。
In conclusion, while CXCR3 provides an advantage to $\mathrm{CD4^{+}}$ T cells for recruitment to the lungs in response to this pulmonary TB vaccine, it is not essential for the development and retention of lung-resident memory $\mathrm{CD4^{+}}$ T cells. This has implications for designing vaccines that aim to induce increased memory $\mathrm{CD4^{+}}$ T cell responses, including $\mathrm{T_{RM}},$ in the lungs at the site of pathogen exposure.
总之，虽然CXCR3为$\mathrm{CD4^{+}}$T细胞响应这种肺结核疫苗向肺部募集提供了优势，但它对于肺部驻留记忆$\mathrm{CD4^{+}}$T细胞（包括$\mathrm{T_{RM}}$）的发育和维持并非必需。这一发现对设计旨在病原体暴露部位（如肺部）诱导增强记忆$\mathrm{CD4^{+}}$T细胞反应的疫苗具有重要启示。
