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B细胞的发育

B细胞发生
B细胞发育的调节
B细胞阳性选择
B细胞阴性选择
B细胞的异质性

B细胞的发生

    免疫系统的独特之处是它能对众多抗原产生应答,包括以前不存在,新近合成的抗原。抗体多样性的特征包括在同一条多肽链上有可变区和恒定区及不同的恒定区连接同一可变区。产生抗体和 T 细胞受体( TCR )多样性的体细胞重组在哺乳动物基因中是独特的,成功合成重链和轻链及表达在细胞膜上是 B 细胞发育所必需及发育各个阶段的标志。
    B 细胞发育是从胎肝开始,在骨髓中继续,下面的表格说明 B 细胞发育的不同阶段的特征。一个 B 细胞在细胞膜表面能表达 μ 和 L 链,然而这时仍为不成熟 B 细胞,此时当它接触到自身抗原后便凋亡,直到它同时表达 IgD 时才为成熟 B 细胞。成熟 B 细胞迁移到外周免疫器官或组织,能被抗原激活而变成抗体分泌的浆细胞或对抗原再次接触产生迅速反应的记忆细胞,未能成功完成成熟发育的 B 将凋亡。

B 细胞发育的阶段

stem
cell

early
pro-B
cell

late
pro-B
cell

large
pre-B
cell

small
pre-B
cell

immature B
cell

mature
B cell

H 链基因
germline

D-J
连接

V-DJ
连接

VDJ
重排

VDJ
重排

VDJ
重排
VDJ
重排
L 链基因
germline
germline
germline
germline
V-J 连接

VJ 重排

VJ 重排

表面 Ig
none
none
none
前 BCR 中有μ链
在细胞质和
表面有μ链

膜 IgM

膜 IgM 和 IgD

RAG 和 TdT 表达
no
yes
yes
no
yes
yes
no
替代 L 链的表达
no
yes
yes
yes
no
no
no

Ig αβ 表达

no
yes
yes
yes
yes
yes
yes
btk*
no
little
yes
yes
yes
yes
yes

膜标志

CD34

CD34
CD45 (B220)
Class II

CD45R
Class II
CD19
CD40

CD45R
Class II
pre-B-R
CD19
CD40

CD45R
Class II
pre-B-R
CD19
CD40

CD45R
Class II
IgM
CD19
CD40

CD45R
Class II
IgM IgD
CD19CD21CD40

* Bruton's tyrosine kinase
    淋巴祖细胞收到来自骨髓基质细胞的信号开始向 B 细胞方向发育。细胞因子诱导 CD34+ 淋巴祖细胞的末端脱氧核苷酸转移酶(TdT)和重组激活酶 (RAG-1 and RAG-2) 的合成。这些细胞在 H 链染色体上进行 D-J 连接,变成早期祖 B 细胞,并且开始表达 CD45 ( B220 )和 MHCII 分子, D-JH 和一个 V 片段连接就完成晚期祖细胞阶段。

    当它们的细胞膜表面表达前 B 细胞受体的时候,即祖 B 细胞变成前 B 细胞,前 B 细胞受体( pre-BCR )由μ HCs 和轻链替代物(ψ LCs )结合而成,轻链替代物与真正的轻链类似,但每一个前 B 细胞都是相同的。信号转导分子 Ig α Ig β同样是前 B 细胞受体复合物中的一部分,膜表面 Ig 的重链胞浆区太短,而不能直接传输抗原结合的信号; Ig α Ig β信号转导分子有免疫受体酪氨酸激活基序( ITAMs ),当抗原与 B 细胞受体( BCR )结合时,它就磷酸化,磷酸化启动胞浆中信号级联反应。该细胞停止 H 链重组和发生产生相同 m 链的克隆增殖。然后成为体积比静止细胞大的细胞,这个阶段称为大前 B 细胞( large pre-B cell )。

    随着进一步增殖,小前 B 细胞的 L 链基因进行 V-J 连接, L 链一旦被成功合成, m 链就表达在细胞膜上,该细胞即被称为不成熟的 B 细胞。不成熟 B 细胞对抗原结合非常敏感, 假如它们在骨髓中与自身抗原结合即凋亡。不能与自身抗原结合,在细胞表面即表达 IgM 又表达 IgD 的将离开骨髓成为成熟的静止 B 细胞。

B 细胞发育的调节

    祖细胞从骨髓基质细胞通过细胞和细胞接触和分泌等方式获得信号。骨髓为 B 细胞的发育提供微环境,基质细胞膜表面的干细胞因子( SCF )和淋巴细胞膜表面的 kit ( CD117 )等黏附分子参与 B 和 T 细胞的发育。 B 和 T 细胞发育的一个重要分泌型细胞因子是由基质细胞分泌的 IL-7 , 它能与发育中的淋巴细胞表面的 IL-7R 结合。通过这些信号启动细胞质中的级联反应,导致发育所需的蛋白表达的改变。当 B 细胞在骨髓中发育的时候,它们从骨髓的外层向中心移动。

    在发育的 B 细胞中,可以发生体细胞重组(导致功能性 H 和 L 链的合成)或由于阅读框移位突变,使终止密码的介入而不发生体细胞重组。发育期间,在一定的时间内,没有产生重排和表达 Ig 的将导致发育 B 细胞凋亡。 B 细胞有两个机会去产生重排重链(母本和父本染色体)和四次机会产生重排轻链(母本和父本、κ和λ位点)。人 B 细胞常常在两条染色体上同时发生 DH 和 DJ 片段的重排。 DH 在任何阅读框下都能读,所以所有的 D-J 重排都能产生。估计只有大约一半的发育 B 细胞产生重链重排,这些克隆扩增的前 B 细胞(大前 B 细胞阶段)能进行轻链重组。

    在小前 B 细胞阶段, V-J 连接的轻链首先是κ链。假如出现重排,κ链被合成,这个细胞就变成一个不成熟的 B 细胞,膜表面表达 IgM(k) BCR 。假如第一次重排没有完成, B 细胞能重复进行几次 V-J 连接;这个过程被称为轻链援救。假如两条染色体的κ基因都没有成功重排,λ基因就被重排,而产生 IgM( λ) BCR 。 假如κ和λ基因都不能重排,这个细胞在骨髓中就发生凋亡。

    在 B 细胞发育期间,体细胞重组和受体表达需要的编码蛋白的基因,在一定时间内开和关。 RAG-1 、 RAG-2 和 TdT 仅在体细胞重组期间表达:即早期和晚期祖 B 细胞及小前 B 细胞阶段。 TdT 常常和重组酶一起关闭。替代 L 链和 Ig α Ig β蛋白一定作为前 BCR 表达在细胞膜表面。

B 细胞的阳性选择

    B 细胞和 T 细胞都在中枢免疫器官中进行阳性和阴性选择。阳性选择是通过抗原受体获得信号即生存,发育中的 B 细胞的阳性选择是发生在前 B 细胞受体与配基结合的时候;阴性选择意谓着与受体结合导致细胞死亡。不成熟的 B 、 T 细胞假如与自身抗原结合就发生阴性选择。

    在基因表达的适当阶段,随着膜前 BCR 和膜 IgM 的表达, B 细胞存活和移动信号出现。有两种实验证明这种叙述。 重排的 H 和 L 链基因被插入小鼠受精卵中而产生转基因小鼠。在转基因小鼠体内,重组的 IgH 和 L 链总是不能与任何其它基因组成 Ig ; 它们在所有的 B 细胞表面表达转基因的 H 和 L 链。转基因鼠的 H 链一直与它们的 L 链重组,反之亦然。因此, VH 和 VL 基因重排的出现,向 B 细胞发出抑制它们进一步重排的信号。

    功能基因被删除的小鼠被称为基因敲除小鼠。分别将小鼠 H 链跨膜区的外显子(使 H 链不能插入膜内)、 Ig α或 Ig β基因(或仅是 ITAMs 部分)或者替代轻链λ 5 和 VpreB 的基因进行敲除,来研究膜表达传递信号的 BCR 复合体的重要性。结果发现,缺少这些蛋白中的任何一个,尽管其他蛋白都能被合成或在 Ig α Ig β缺乏 ITAMs 的细胞膜上有完整的前 BCR 表达,都会阻碍 B 细胞的发育。 替代轻链λ 5 类似于λ链的恒定区,但是由不同基因编码的。λ 5 以非共价键与一个 VpreB (类似 Ig V 区)结合。 既然前 B 细胞表达许多不同的 VH 区,假设所有前 B 细胞的 VpreB 相同,它与一条轻链结合作为前 B 细胞分裂然后开始轻链重组的信号。同时也发出关闭其他未确认的轻链重组的信号。

    体细胞重组导致在各自 B 细胞内 H 和 L 链的等位基因排除,即每一个 B 细胞只有一个 H 链基因和一个 L 链基因重组。在杂合子中,每一个等位基因表现在一半的 B 细胞和一半的血清 Ig 分子中。轻链同样显示同型排除,即一个细胞或分子只有κ或λ链。κ或λ链不可能同时出现在一个 B 细胞或血清 Igs 上。在人类,κ链比λ链多 65% 到 35% 。 在小鼠,血清 Ig95% 是κ链,而猫 95% 是λ链。κ和λ的比率反映了每一同种型中 V 区片段的相对数量和它们重组入功能性 L 链的相对效率。

    一旦 B 细胞离开骨髓,它的生存似乎依赖于二级淋巴组织的淋巴滤泡中进一步信号的刺激。新生的 B 细胞和较老的 B 细胞之间对这些信号的竞争,可能维持着 B 细胞的动态平衡。例如已有实验显示,注射的转基因 B 细胞的生存依赖于宿主正常 B 淋巴细胞通过照射清除。

B 细胞的阴性选择

    当它们结合多价配基时,仅表达 IgM 的 B 细胞被清除或没有活性,而不象成熟 B 细胞通过 BCR 的交联被激活。在骨髓中,结合多价自身抗原导致 B 细胞凋亡和克隆清除;结合可溶性自身抗原不能使 B 细胞死亡;这个细胞能进入外周血中,表达 IgD 有很少的 IgM 。 这些细胞是无反应性的;它们不能对抗原应答,寿命较短。不能与自身抗原结合的细胞表达正常水平的 IgM 和 IgD ;假如它们成功进入淋巴滤泡,就能生存几周直到它们遇到特异性抗原或者死亡。

    尽管许多自身特异性 B 细胞被克隆清除,但一些 B 细胞可以进行进一步的体细胞重组而生成新的没有自身特异性的 VH 和 VL 复合体。有实验显示,通过受体编辑改变它们的特异性能够挽救一些自身反应性 B 细胞的命运。在许多种动物中, Ig 的胚系( germline )多样性是不存在或很低的。仅有一个或少量的功能性 V 、 D 和 J 片段用于重组,以至于所有不成熟 B 细胞有同样的抗原特异性和结合自身抗原。 不成熟 B 细胞结合自身抗原,为细胞分裂发出信号;并且在分裂期间, DNA 与邻近的假基因(包含停止密码的基因片段)互换从而改变 V 区核苷酸序列。通过基因转变产生了 Ig V 区的多样性。一旦细胞不再结合自身抗原,它们成熟并进入外周血。

B 细胞的异质性

    在胚胎期间,骨髓干细胞发育成不同特征的 B 细胞;被称为 B-1 B 细胞, B-1B 细胞有膜 CD5 ,它们能自我更新,即它们在外周淋巴组织中分裂成更多和它们一样的成熟静止 B 细胞。一般来说, B-2 细胞仅在对抗原应答时增殖,而成为在外周血中的记忆或浆细胞;更多的静止 B-2 细胞必须由骨髓中的祖细胞补充。 B-1 BCR 的多样性比 B-2 细胞少, B-1 BCR 仅从一些 Ig 基因片段中产生,在基因片段中间没有另外的氨基端核苷酸,并且主要针对普通细菌的碳水化合物抗原。 B-1 细胞主要分泌 IgM 和有很少的体细胞高度突变, 因为它们对多种抗原产生应答和与许多抗原结合的亲和力比较低, B-1 细胞和它们分泌的抗体被称为多反应性。 在没有免疫的小鼠发现有许多被 B-1 细胞产生的 IgM , 出生后 B-1 产生 Ig 比出生前具有更多的多样性,但没有 B-2 细胞那么多的多样性。最后,骨髓干细胞停止产生 B-1 细胞。 与 T 细胞发育早期产生γδ T 细胞类似。

    随着 B 细胞发育的不同阶段, B 细胞改变着居住地,每个部位都为它在此阶段的发育提供适宜的微环境。在骨髓中,也只有在骨髓中干细胞才产生淋巴祖细胞和祖 B 细胞。 随着它们的成熟,发育中的 B 细胞向骨髓中心移动,成熟的 B 细胞离开骨髓,通过选择素和血管内皮细胞表面的粘着素结合进入外周淋巴组织,通过 T 细胞依赖区进入 B 细胞依赖区 ( 滤泡 ) 。肠道集合淋巴结、扁桃体和阑尾主要由大滤泡组成。在黏膜相关淋巴组织( MALT )滤泡的微环境中,给 B 细胞发出产生 IgA 的信号,而在淋巴结和脾脏给 B 细胞发出产生 IgG 的信号。

    在滤泡中, B 细胞遇到抗原并且受到来自生发中心( germinal centers ) T 细胞区中的 T 细胞的适当帮助,它们迅速增殖、进行体细胞高度突变和选择具有高亲和力受体的 B 细胞。主要在淋巴结的髓索中、脾脏的红髓、骨髓(主要 IgG 分泌的浆细胞)和黏膜固有层中发现,抗体分泌的浆细胞,短命的不能通过滤泡,长命的在滤泡中进行体细胞高度突变和抗体类型转换。记忆性 B 细胞主要存在于脾脏的边缘区、淋巴结的 sub-capsular sinus 、肠道集合淋巴结的肠上皮下和扁桃体的隐窝( crypt );在血中很少被发现。

    在 B 细胞发育的不同阶段,都可发生肿瘤,这些 B 细胞肿瘤有助于免疫学家对 B 发育的认识。每一种类型都有它的 Ig 基因重组特征和归巢特性。 几乎每一个病例都是单克隆的,即由单个 B 细胞发育成的癌细胞。在一些 B 细胞肿瘤中发现, DNA 的易位导致癌基因的被激活,是引起肿瘤的原因。

其它网络资源
Practice Quiz

Pick the one best answer to each question by clicking on the letter of the correct choice.

1. B cell differentiation begins with the expression of

a. membrane m chain + surrogate L chain.

b. membrane IgD.

c. Membrane IgM.

d. germline IgM.

e. RAG-1, RAG-2 and TdT.

2. Bone marrow stromal cells

a. are important because they provide physical support for B cells (hence, their name from the Greek word for mattress).

b. are present only in the center of the marrow.

c. present foreign antigen to B cells to stimulate somatic hypermutation .

d. present self antigen on self MHC to B cells for negative selection.

e. secrete cytokines such as IL-7 that signal developing B cells to divide and differentiate.

3. Cell adhesion molecules (CAMs)

a. are found only on bone marrow stromal cells.

b. are specific receptors for cytokines that promote cell-cell binding.

c. function primarily to hold developing B cells in one location until they are fully developed.

d. signal developing B cells to divide and differentiate.

e. signal developing B cells to die because they have bound self.

4. The developmental step that commits a cell to the B lineage is

a. expression of both membrane IgM and IgD.

b. expression of membrane m chain.

c. expression of recombinase enzymes.

d. joining of a VH gene segment to a DH gene segment.

e. joining of a DH gene segment to a JH gene segment

5. Which statement about B cell development is FALSE?

a. Cells which fail to synthesize and express m chains usually die.

b. Each DNA joining event in Ig genes has a 67% probability of resulting in a nonproductive rearrangement.

c. The earliest developing B cell which could be stained with FITC-anti-m chain would be a pro B cell.

d. The earliest developing B cell which could be stained with FITC-anti- k chain would be an immature B cell.

e. The enzyme which can add nucleotides not encoded in the DNA to Ig genes during recombination is TdT.

6. Once H chain genes have been productively rearranged and expressed on the pre-B cell membrane, the next event to occur in the cell is

a. death of cells binding self antigen.

b. expression of membrane IgD.

c. expression of membrane of IgM.

d. proliferation of the pre-B cells.

e. somatic recombination of light chain genes.

7. In a productive rearrangement of Ig DNA, what is produced must be

a. a functional membrane Ig protein chain.

b. a loop of DNA that is then removed.

c. an mRNA for H or L chain.

d. a shorter piece of DNA.

e. successful antibody secretion in response to antigen.

8. Proliferation of large pre-B cells

a. is part of clonal selection.

b. makes the pre-B cells more susceptible to apoptosis following self antigen binding

c. results in production of many B cells with the same antigen specificity.

d. results in the production of many B cells with the same VH chain but different antigen specificities due to different VL regions.

e. requires the presence of RAG-1 and RAG-2.

9. Which of the following statements is TRUE?

a. Isotypic exclusion on individual B cells pertains to expression of a single heavy chain isotype on each mature naive B cell.

b. Pre-B cells must receive a signal from specific antigen binding to pre-B receptor before they can proceed to the next stage in development.

c. Membrane m chain is always expressed with Iga and Igb

d. Membrane m chain is always expressed with VpreB and l5.

e. Transgenic mice for recombined H and L immunoglobulin genes have germline H and L genes in non-B cells.

10. Light chain rescue

a. allows self-specific B cells to repeat somatic recombination of light chain gene segments.

b. is a signal received through binding to the surrogate light chain that rescues the developing B cell from death.

c. results from multiple V-J joining events on a single chromosome until productive rearrangement of light chain occurs or all J segments have been recombined.

d. signals the developing B cell through IgaIgb to begin recombination of light chain gene segments.

e. None of the above is true.

11. Regulatory nucleotide sequences in the DNA that control Ig protein synthesis are

a. inducers and promoters.

b. initiation sites and enhancers.

c. promoters and enhancers.

d. promoters and switch regions.

e. recombination signal sequences and promoters.

12. Transgenic mice for BCR

a. are given a completely new set of Ig genes.

b. express the recombined H chain gene on every cell in the body.

c. have germline H chain gene segments in all their cells except B cells.

d. suppress their own H chain V-D-J recombination if they have been given a recombined H chain gene.

e. synthesize Ig with the same amount of diversity as non-transgenic mice.

13. If the Ig V regions encoded in the transgenes were specific for self MHC, the transgenic mice

a. might be able to produce low numbers of non-self-specific B cells through receptor editing.

b. would produce normal numbers of B cells because MHC is not expressed in the marrow.

c. would die of autoimmunity.

d. would fail to produce any B cells.

e. would not die of autoimmunity because only T cells bind MHC.

14. In general, a knock-out mouse

a. does not show allelic exclusion of Ig.

b. has a mutant TdT that removes (knocks out) N nucleotides instead of inserting them during somatic recombination.

c. has no Ig gene segments.

d. has a normal gene replaced with a nonfunctional gene for the same protein.

e. is often used to produce monoclonal antibody.

16. Knock-out mice which have m gene segments from which the membrane domain has been removed

a. cannot synthesize any m chain.

b. can splice the Cm segments to the membrane segment for d and express normal amounts of membrane m chain.

c. have normal pro-B cells but no later stages of developing or mature B cells.

d. have normal pro-B cells and pre-B cells but no mature B cells.

e. have no cells with rearranged Ig genes.

17. B cells which express Iga with a truncated cytoplasmic domain (lacking the ITAMS)

a. become "stuck" in the immature B cell stage of development.

b. can express pre-B cell receptor but not proceed to divide and then recombine light chain genes.

c. develop normally since Igb has its ITAM sequences.

d. express both IgM and IgD but cannot be activated by antigen.

e. require more antigen for activation.

18. The ability of an individual B cell to express only one H chain allotype is called

a. allelic exclusion.

b. co-dominant expression.

c. isotypic exclusion.

d. nonproductive rearrangement.

e. survival of the fittest.

19. Once they leave the marrow, in order to survive mature naive B cells must enter the

a. circulation

b. lymph microenvironment.

c. primary lymphoid follicles.

d. secondary lymphoid follicles.

e. thymus.

20. Negative selection of B cells occurs when

a. immature B cells bind self antigen and undergo apoptosis.

b. immature B cells fail to bind self MHC and die.

c. lymphoid progenitor cells become committed to becoming T cells and leave the marrow for the thymus.

d. mature B cells fail to bind antigen in the lymphoid follicles and die.

e. pre-B cells bind ligand with their pre-B receptor and stop rearranging H chain genes.

21. A mature lymphocyte which has specific antigen receptors but cannot respond to that antigen is called

a. anergic.

b. apoptotic.

c. deleted.

d. lazy.

e. memory.

22. Immature B cells which bind soluble self antigen

a. become apoptotic.

b. escape clonal deletion and can potentially cause autoimmune disease in adults.

c. go on to express normal levels of IgM and IgD but cannot respond to self antigen.

d. undergo clonal deletion in the bone marrow.

e. usually cannot enter the primary lymphoid follicles and soon die in the periphery.

23. Immature B cells from inbred mouse strain q (expressing the q allele of every MHC molecule: Dq, Kq, Lq, IAq, and IEq) which also express transgenic H and L chains specific for H-2 IAs (the s allele of Class II MHC IA) will undergo clonal deletion when

a. exposed to macrophages from an "s" strain mouse (expressing the "s" allele of every MHC molecule).

b. exposed to T cells from an s strain mouse.

c. exposed to their own macrophages.

d. exposed to their own T cells.

e. Both 1 and 2 are correct.

24. Chickens have very few functional V, D, and J gene segments and therefore

a. are very susceptible to disease.

b. have many self-specific lymphocytes and usually die of autoimmune disease.

c. insert diverse sequences from VH pseudogenes into their H chains while B cells are immature.

d. make mature B cells which are all specific for the same antigen.

e. use gene conversion to introduce diversity into Ig gene sequences as B cells divide in response to foreign antigen.

25. The B cells with the longest life span are the

a. anergized B cells.

b. immature B cells.

c. mature naive B cells.

d. memory cells.

e. plasma cells.

26. Receptor editing

a. allows self-specific B cells to repeat somatic recombination.

b. is responsible for allelic exclusion of Ig on individual B cells.

c. kills self-specific B cells before they leave the marrow.

d. occurs during the small pro-B cell stage of development.

e. occurs only for light chains of self-specific B cells.

27. B-1 B cells are more _____________ than conventional B (B-2) cells.

a. antigen-specific

b. high affinity

c. likely to be protein-specific

d. numerous in adults

e. polyreactive

28. The antibody secreted by B-1 B cells often

a. binds common bacterial carbohydrates.

b. has a high affinity for antigen.

c. is IgG.

d. neutralizes common viral receptor proteins.

e. neutralizes self antigens.

29. As cells become mature naive B cells, they

a. become more antigen-independent.

b. leave the marrow and never return.

c. stay in one location and wait for antigen and T cells to come to them.

d. stay in the same locations as T cell to be able to receive helper signals.

e. use CAMs to enter the appropriate lymphoid organ locations.

30. A germinal center is where B cells

a. become mature.

b. divide in response to antigen.

c. first bind antigen.

d. secrete antibody.

e. undergo somatic recombination.

31. If you wanted to check for the presence of memory B cells from your vaccine subjects by taking some of their B cells and re-stimulating them in vitro (outside the body) with antigen, the best place to get some memory cells would be from

a. blood.

b. bone marrow.

c. lymph nodes.

d. spleen.

e. tonsils.

32. Multiple myeloma is a cancer which has arisen from a

a. B-1 B cell.

b. lymphoid progenitor.

c. mature B cell.

d. plasma cell.

e. pre-B cell.

33. If a B cell tumor is monoclonal, it can be differentiated from normal B cells of the same person by its unique membrane Ig

a. allotype.

b. H chain.

c. idiotype.

d. isotype.

e. L chain.

34. An oncogene is a

a. gene for a virus protein that causes cancer.

b. gene for tumor antigens on B cells.

c. mutated gene that causes cancer.

d. normal gene that can cause cancer if its normal function is disrupted.

e. translocated Ig gene.

Problems

1. I have goat anti-rabbit ga and gb, antibodies specific for the "a" and "b" allotypic determinants on the rabbit g heavy chain. I also have a rabbit heterozygous for the "a" and "b" alleles of g chain). The goat anti- ga is tagged with FITC; the goat anti gb is tagged with PE. How can I prove with these reagents that IgG is allelically excluded on the rabbit's B cells? Draw a flow diagram illustrating the staining of rabbit B cells with the FITC-anti-ga and the PE-anti-gb.

2. Cells were removed from the bone marrow of an adult mouse and incubated with FITC-anti mouse m chain and PE-anti mouse d chain. Draw the flow cytometric representation of the data. What cell types are in each quadrant of the graph: stem cells, lymphoid progenitors, myeloid progenitors, pro-B cells, pre-B cells, immature B cells, mature B cells, neutrophils, macrophages? What would be the pattern of fluorescence you would see if you stained spleen cells with the same antibodies? Are the kinds of cells in each quadrant the same? HINT: consider all the kinds of leukocytes which are found in the spleen. How would this pattern change if you used mice lacking RAG-1 and RAG-2?

3. Make a line drawing of germline DNA and DNA from an early pro-B cell, a late pro-B cell, a large pre-B cell, and an immature B cell for both H and k genes. Write a rule that would allow you to identify each of line drawings:

A sequence must be germline H chain DNA if...

A sequence must be H chain DNA in an early pro-B cell if ...

A sequence must be H chain DNA in a late pro-B cell if ...

A sequence must be H chain DNA in an immature B cell if ...

A sequence must be germline L chain DNA if...

A sequence must be L chain DNA in an early pro-B cell if ...

A sequence must be L chain DNA in an late pro-B cell if ...

A sequence must be L chain DNA in an immature B cell if ...

Would the sequence be any different in a mature B cell? In a plasma cell secreting IgG1 in the lymph nodes? In a plasma cell secreting IgA2 in the MALT?

4. Starting with two cell lines from a mouse, one with cytotoxic activity and one with helper activity, how could you prepare and purify a rabbit anti-mouse CD4?

5. A transgenic mouse was constructed by inserting recombined H and L chain genes encoding an antibody to mouse H-Y antigen, which is expressed on the membrane of all cells of male but not female mice. Predict what B cell development will occur in male and in female transgenic mice. Describe or draw the flow cytometry data from spleen cells of male and of female mice stained with FITC-anti-m and PE anti-idiotype (for the transgenic Ig).

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