Localization of B and T cell

Localization of B and T cell

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What does localization of B-Cell mean??
"Localization of B and T cell in allergens may not coincide". What does this statement mean?
(I have not studied biology since last 8 years and now I am going through it because I need it for my research. So if someone can describe it in simple language it would be very helpful)

I think the reason you are having trouble discerning the statement is because it doesn't really make much sense, nor is it in my opinion sufficiently explained in the paper. The sentences preceding the it give a little explanation as to what the author may be getting at:

So far, 1500 allergenic structures have been identified. Online allergen databases and allergy prediction tools are being used to find cross-reactivity between known allergens. Localization of B and T cells in the allergen may not coincide.

During an exaggerated immune response (allergy) the lymphocytes (B and T) will localize (travel or accumulate at) to the site of the antigen (allergen).

If I could guess I would say the author is stating that data has shown that no immune response is taking place (data generated by assessing the accumulation of lymphocytes near the allergen), despite the fact that these online allergen predictors say that an immune response should be generated.

The statement in my opinion moves to discredit these online allergen predictors.

I did look over some of the rest of the manuscript, and found two other ambiguities within.

The author does cite another paper in your sentence that may shed more light on this:

An overview of it provided little clarity however.

I fear that only a crystal ball or this question addressed to the senior author may answer your question with complete certainty:

Dr. Rajat Kumar De Professor Machine Intelligence Unit Indian Statistical Institute 203 Barrackpore Trunk Road, Kolkata 700108, India.

"Localization of B and T cell in allergens may not coincide". That sentence is written poorly, it confuses more than inform.

What the author means is: The epitopes that B cells and T cells can recognize are not the same in one particular allergen. We have to remember that almost all allergens are proteins and proteins are made up of aminoacids, and that's what make up epitopes. For one same particular structure or say, an allergen, the T cell can only recognize LINEAR epitopes whereas the B cell can recognize LINEAR or CONFORMATIONAL epitopes (that is, they recognize a 3d-like structure and not just a sequence of aminoacids).

What I always tell my students so they can grasp the concept is that the T cells are able to "see" a photograph of a chair to recognize it. While a B cell can do that and even more it can actually "see" a chair for what it is.

Phosphatidic acid-dependent localization and basal de-phosphorylation of RA-GEFs regulate lymphocyte trafficking

Lymphocytes circulate between peripheral lymphoid tissues via blood and lymphatic systems, and chemokine-induced migration is important in trafficking lymphocytes to distant sites. The small GTPase Rap1 is important in mediating lymphocyte motility, and Rap1-GEFs are involved in chemokine-mediated Rap1 activation. Here, we describe the roles and mechanisms of Rap1-GEFs in lymphocyte trafficking.


In this study, we show that RA-GEF-1 and 2 (also known as Rapgef2 and 6) are key guanine nucleotide exchange factors (GEF) for Rap1 in lymphocyte trafficking. Mice harboring T cell-specific knockouts of Rapgef2/6 demonstrate defective homing and egress of T cells. Sphingosine-1-phosphate (S1P) as well as chemokines activates Rap1 in a RA-GEF-1/2-dependent manner, and their deficiency in T cells impairs Mst1 phosphorylation, cell polarization, and chemotaxis toward S1P gradient. On the other hand, B cell-specific knockouts of Rapgef2/6 impair chemokine-dependent retention of B cells in the bone marrow and passively facilitate egress. Phospholipase D2-dependent production of phosphatidic acid by these chemotactic factors determines spatial distribution of Rap1-GTP subsequent to membrane localization of RA-GEFs and induces the development of front membrane. On the other hand, basal de-phosphorylation of RA-GEFs is necessary for chemotactic factor-dependent increase in GEF activity for Rap1.


We demonstrate here that subcellular distribution and activation of RA-GEFs are key factors for a directional movement of lymphocytes and that phosphatidic acid is critical for membrane translocation of RA-GEFs with chemokine stimulation.


Dynein Collaborates with NMII to Move the Centrosome.

To investigate the role of dynein, we transduced primary CD4 + T-cell blasts with shRNA against the dynein heavy chain (DynHC) together with a construct encoding RFP-labeled centrin, a centrosomal marker ( Fig. 1 A and B ). The T cells expressed the 5C.C7 TCR, which recognizes the moth cytochrome c88� (MCC) peptide bound to the class II MHC molecule I-E k . Suppression of DynHC markedly dispersed the Golgi apparatus, indicative of impaired dynein function (Fig. S1). We then assessed centrosome polarization by imaging fixed conjugates formed by T cells and antigen-loaded CH12 B cells ( Fig. 1 BD ). As expected, control T cells expressing nontargeting shRNA displayed robust centrosome polarization, characterized by a “polarization index” parameter close to zero. This response was abrogated by nocodazole, which depolymerizes the microtubule cytoskeleton, and by phorbol myristate acetate (PMA), which blocks centrosome reorientation by eliciting unpolarized DAG signaling (4). Surprisingly, T cells lacking dynein displayed only a minor polarization defect that in some experiments failed to reach statistical significance. Similar results were obtained using a recently described small-molecule dynein inhibitor, ciliobrevin D ( Fig. 1E ), which targets the dynein motor domain (17). Hence, loss of dynein protein or dynein function only partially blocked centrosome reorientation, implying the existence of compensatory mechanisms.

Dynein and NMII collaborate during centrosome polarization in T cell𠄺PC conjugates. (AC) T-cell blasts (5C.C7) expressing the indicated GFP-marked shRNAs together with centrin-RFP were mixed with antigen-loaded CH12 cells and imaged after fixation. Cells were treated with 5 ng/mL PMA, 33 μM nocodazole, 50 μM blebbistatin, or vehicle control (DMSO) as indicated. (A) Validation of DHC shRNA knockdown by immunoblot, with β actin serving as a loading control. NT, nontargeting shRNA control. (B) Representative fluorescence images are shown overlaid onto their corresponding bright-field images. (Scale bars: 10 μm.) (C) Schematic showing the calculation of polarization index. (D) Quantification of polarization index in fixed conjugates (n ≥ 44 conjugates per condition). (E) Centrosome polarization in fixed conjugates treated with DMSO vehicle, 5 ng/mL PMA, 50 μM ciliobrevin D, 50 μM negative control compound for ciliobrevin D (compound 2), 50 μM blebbistatin, or 50 μM ciliobrevin D in combination with 50 μM blebbistatin (n ≥ 45 conjugates per condition). Error bars in D and E denote SEM. P values were calculated using the Mann–Whitney test. ***P < 0.001 **P < 0.01 *P ≤ 0.05 ns, P > 0.05.

Because NMII has been implicated in polarity induction in adherent cell types (10, 11), we investigated whether it might contribute to centrosome reorientation in T cells. Blebbistatin, a specific inhibitor of the myosin II motor, induced a small polarization defect, similar in magnitude to that induced by dynein deficiency alone ( Fig. 1 BE ). However, combining blebbistatin with either DynHC shRNA or ciliobrevin D profoundly inhibited centrosome polarization, with average polarization indices comparable to nocodazole- and PMA-treated cells. TCR-induced Erk phosphorylation was unaffected by inhibition of dynein or NMII, implying that the polarization phenotypes we observed did not result from impaired TCR signaling (Fig. S2A). These results indicated that NMII and dynein operate in a partially redundant manner to move the centrosome toward the IS.

To closely examine the interplay between dynein and NMII and to correlate polarization responses with other signaling events, we used a previously described TCR-photoactivation and imaging assay (18). T cells are attached to coverslips coated with a photocaged version of their cognate pMHC. Subsequent irradiation of a micron-sized area beneath the T cell with UV light induces localized TCR activation, establishing an IS-like region within the T cell–glass interface. DAG and nPKCs typically accumulate in this region after � s, with centrosome reorientation following 10� s later (4). This system enables us to trigger TCR-dependent centrosome reorientation and monitor associated responses with high spatiotemporal resolution.

Suppression of DynHC resulted in a small, but detectable, defect in centrosome reorientation to the UV-irradiated region ( Fig. 2 A and B ). T cells lacking DynHC also displayed a significant reduction in maximum centrosome speed, suggesting that the capacity to move the centrosome was impaired ( Fig. 2C ). Similar defects were observed after blebbistatin treatment ( Fig. 2 AC ), consistent with a role for NMII in the process. shRNA-mediated suppression of MyH9, the only NMII heavy chain expressed in T cells, also inhibited centrosome reorientation, although to a lesser extent than blebbistatin (Fig. S3A). This likely reflected suboptimal knockdown by the MyH9 shRNA (Fig. S3B). Importantly, simultaneous application of DynHC shRNA and blebbistatin inhibited polarization responses to a much greater extent than either treatment alone. Movement of the centrosome toward the irradiated region was essentially abrogated ( Fig. 2 A and B ), and maximum centrosome speed was reduced more than twofold relative to control T cells ( Fig. 2C ). Knockdown of DynHC did not affect TCR-induced DAG production, nor did blebbistatin alter recruitment of PKCθ, indicating that early TCR signaling was intact (Fig. S2 B and C). These data indicate that dynein and NMII collaborate to move the centrosome downstream of polarized nPKC activation.

Dynein and NMII collaborate to polarize the centrosome in response to TCR photoactivation. Centrosome polarization in T cells treated with nontargeting (NT) shRNA or shRNA against DynHC (DHC), with or without 50 μM blebbistatin. (A) Representative time-lapse montages, with the time of UV irradiation indicated by yellow text. Yellow circles denote the irradiated region in each experiment. Time is indicated as minutes:seconds above the montages. (Scale bars: 5 μm.) (B) Average distance between the photoactivated region and the centrosome over time, with UV irradiation indicated by a purple line. (C) Maximum speed of centrosome movement (n ≥ 34 cells per sample). Error bars denote SEM. P values were calculated using Student t test. ***P < 0.001 **P < 0.01 *P ≤ 0.05 ns, P > 0.05.

Reciprocal Localization of NMII and Dynein During Centrosome Reorientation.

To explore how NMII influences centrosome polarization, we monitored its localization in photoactivation experiments using T cells expressing GFP-labeled MyoRLC. NMII formed transient, filamentous clusters beneath the plasma membrane ( Fig. 3A ) that could be visualized by total internal reflection fluorescence (TIRF) microscopy. Localized TCR photoactivation altered this pattern by suppressing the formation of new NMII clusters in the irradiated region ( Fig. 3 A and B and Movie S1). This generated asymmetry in the NMII distribution, because clusters continued to form in the membrane behind the centrosome as it reoriented ( Fig. 3B ). Close analysis of individual steps in centrosome movement revealed a marked correlation between the instantaneous speed of the centrosome and the MyoRLC intensity differential measured along the direction of movement ( Fig. 3C ). Thus, larger speeds were associated with higher accumulation of MyoRLC behind the centrosome and greater depletion in front of it. In addition, cross-correlation analysis indicated that loss NMII from the irradiated region preceded centrosome reorientation by 27.0 ± 5.9 s. Hence, NMII remodeling occurred at the right time to influence the polarization response.

Reciprocal localization of NMII and dynein after TCR activation. Photoactivation experiments were performed using 5C.C7 T cells expressing MyoRLC-GFP together with either centrin-RFP (AC) or DynIC-RFP (D and E). MyoRLC-GFP and DynIC-RFP were imaged using TIRF microscopy, and centrin-RFP was imaged with epifluorescence. (A and D) Representative time-lapse montages, with the time of UV irradiation indicated by yellow text. Yellow circles denote the irradiated region in each experiment. Arrowheads in A show MyoRLC-GFP clusters. Time is indicated as minutes:seconds at the top of each image. (Scale bars: 5 μm.) (B and E) Quantification of MyoRLC and DynIC dynamics (n ≥ 10 cells for each curve). Exclusion of MyoRLC from the irradiated region (B and E) and recruitment of DynIC to the irradiated region (E) are shown as 㥏/F, which is normalized background corrected mean fluorescence intensity (MFI). MyoRLC rearrangement was also assessed by calculating the MFI ratio between the back and the front of the T cell (B) (see also SI Materials and Methods). (C) Correlation between centrosome step size and differential accumulation of MyoRLC around the centrosome, calculated at each time point by subtracting the MyoRLC MFI in front of the centrosome from the MFI behind centrosome. Data are sorted based on the step size of the centrosome at the same time point (n = 10 cells see also SI Materials and Methods). Error bars indicate SEM.

Dynein is recruited to the region of TCR stimulation before the centrosome (4). This recruitment response can be monitored by TIRF imaging in live cells using fluorescently labeled dynein subunits including the intermediate chain (DynIC), the light intermediate chain (DynLIC), and the TcTex light chain (DynLC). Using T cells expressing GFP-labeled MyoRLC together with RFP-labeled DynIC, we found that dynein and NMII adopted reciprocal configurations during polarization responses ( Fig. 3 D and E and Movie S2). Whereas dynein was recruited to the irradiated zone, myosin clustered in regions lacking dynein. This marked anticorrelation was detectable both before and after TCR stimulation, suggesting that the reciprocal localization of NMII and dynein is not established by TCR signaling but is merely harnessed by it (Fig. S4A). NMII depletion preceded dynein accumulation in the irradiated region by 24.8 ± 11.5 s, indicating that NMII is reorganized before dynein in this pathway. Taken together with the functional experiments described above, these results support a model whereby dynein “pulls” on the microtubule network from the front while NMII “pushes” it from behind.

It has been reported that NMII accumulates at the IS in T cell𠄺PC conjugates (13), which is seemingly incompatible with the idea that it could influence centrosome polarization from the rear. To investigate this issue, we imaged T cells expressing both MyoRLC-GFP and centrin-RFP together with APCs (Fig. S4B). Although we did occasionally observe synaptic accumulation of NMII, it occurred in less than half of the conjugates we examined (5/14) and, in all cases, lasted less than 3 min. Interestingly, we also observed transient NMII puncta forming on the sides and backs of activated T cells during centrosome reorientation (white arrowheads in Fig. S4B). Although we cannot say with certainty that these puncta are identical to the NMII clusters observed in photoactivation experiments, they formed at the right place and at the right time to be involved in the polarization response. Hence, the localization of NMII in T cell𠄺PC conjugates is not inconsistent with our proposed model.

NPKCs Regulate the Localization of NMII and Dynein.

Next, we investigated whether TCR-induced NMII and dynein remodeling require localized DAG accumulation. PMA, which masks the effects of DAG gradients by inducing unpolarized DAG signaling, abrogated both NMII and dynein remodeling in photoactivation experiments ( Fig. 4 A and B ). This implied a crucial role for localized DAG in the regulation of both motors.

NMII and dynein asymmetry is regulated by nPKC activity downstream of DAG. (A and B) T cells (5C.C7) expressing either MyoRLC-GFP (A) or DynIC-GFP (B) were photoactivated and imaged in the presence of 5 ng/mL PMA or vehicle control. Quantification of NMII clearance and dynein recruitment are shown (n ≥ 8 cells per sample). (CE) T cells (5C.C7) derived from PKCθ −/− (θKO) mice were transduced with MyoRLC-GFP together with the indicated shRNAs and used for photoactivation experiments. (C) Validation of shRNA knockdown by immunoblot, with β actin serving as a loading control. NT, nontargeting shRNA control. (D) Representative time-lapse montages comparing MyoRLC distribution in θKO cells with or without PKCη and PKCε. The time of UV irradiation is indicated by yellow text, and the irradiated region is denoted by yellow circles. (Scale bars: 5 μm.) (E) Quantification of NMII clearance from the irradiated region in θKO cells with or without PKCη and PKCε (n ≥ 10 cells for each sample). MyoRLC and DynIC dynamics were quantified as in Fig. 3 , with purple lines indicating UV irradiation. Error bars denote SEM.

DAG promotes centrosome reorientation by recruiting PKCε, PKCη, and PKCθ to the IS (5). To determine whether these kinases are required for the reciprocal distribution of dynein and NMII, we monitored the dynamics of MyoRLC-GFP and DynLC-GFP in T cells lacking various combinations of nPKCs. Simultaneous suppression of PKCη and PKCε, which function redundantly in this pathway (5), did not affect clearance of NMII from the irradiated region ( Fig. 4C and Fig. S5A). Likewise, cells derived from PKCθ −/− mice did not exhibit a significant defect in NMII remodeling ( Fig. 4 D and E ). Suppression of PKCη and PKCε in a PKCθ knockout background, however, completely abrogated NMII dynamics ( Fig. 4 D and E and Fig. S5B). Dynein accumulation at the irradiated region was also blocked in cells lacking PKCη, PKCε, and PKCθ (Fig. S5 C and D). These results indicated that nPKC activity is essential for the relocalization of both NMII and dynein. Consistent with this interpretation, relatively low concentrations (500 nM) of the broad specificity PKC inhibitor G� suppressed TCR-induced NMII depletion and dynein accumulation (Fig. S5 E and F). Taken together, these results demonstrate that PKCη, PKCε, and PKCθ function redundantly to regulate NMII and dynein during centrosome polarization.

To assess whether PKC activity is sufficient to induce NMII remodeling, we used TIRF microscopy to monitor the effects of acute PKC stimulation on the localization of MyoRLC. PMA, which globally activates PKCs, induced the dramatic dispersion of cortical NMII fibers within seconds ( Fig. 5A and Movie S3). We quantified this reorganization by calculating the SD of MyoRLC fluorescence in each cell, which reflects the degree of its clustering ( Fig. 5B ). Acute inhibition of PKC activity with G� had the opposite effect, enhancing NMII cluster formation beneath the membrane ( Fig. 5 A and C and Movie S4). Simultaneous addition of both PMA and G� also promoted clustering, indicating that cluster suppression by PMA requires PKC activity ( Fig. 5 A and D and Movie S5). These results strongly suggest that PKC-mediated phosphorylation of NMII promotes its dissociation from membrane complexes. Interestingly, acute addition of PMA or G� had no effect on the cortical distribution of dynein (Fig. S6), implying that global stimulation of PKCs is insufficient to induce dynein recruitment. Hence, whereas PKC activation is sufficient to suppress NMII clustering, the regulation of dynein is likely to be more complex.

Acute activation or inhibition of PKC activity induces NMII remodeling. T cells (5C.C7) expressing MyoRLC-RFP were imaged in TIRF and treated with 5 ng/mL PMA or 500 nM G� as indicated during time-lapse acquisition. (A) Representative time-lapse montages, with addition of reagents indicated by the red line. (Scale bars: 5 μm.) (BD) Clustering of MyoRLC at the membrane was quantified by calculation of the SD of the fluorescence signals for each cell (n = 10 cells for each curve see also Materials and Methods). The time of reagent addition is indicated by the gap in each curve. Error bars denote SEM.

PKC Phosphorylation Sites Within MyoRLC Are Required for NMII Suppression.

PKC-mediated phosphorylation of MyoRLC at Ser1 and Ser2 has been reported to inhibit NMII function (19 �). To investigate the importance of these phosphorylation events for centrosome polarization, we analyzed the localization of a MyoRLC construct [MyoRLC(S1AS2A)], the N-terminal phosphorylation sites of which were mutated to Ala. MyoRLC(S1AS2A) accumulated in clusters beneath the plasma membrane that were morphologically similar to structures containing wild-type MyoRLC ( Fig. 6A ). However, whereas photoactivation consistently suppressed clustering of wild-type MyoRLC in the irradiated region, depletion of MyoRLC(S1AS2A) was markedly impaired ( Fig. 6 A and B ).

nPKC recruitment and MyoRLC phosphorylation is associated with NMII remodeling. (A and B) TCR-photoactivation experiments were performed using 5C.C7 T cells expressing either wild-type MyoRLC-GFP or MyoRLC(S1AS2A)-GFP. (A) Representative time-lapse montages, with the time of UV irradiation indicated by yellow text. Yellow circles denote the irradiated region. (B) Quantification of MyoRLC clearance from the irradiated region (n ≥ 10 cells for each sample). (CF) TCR-photoactivation experiments were performed using 5C.C7 T cells expressing MyoRLC-GFP together with either PKCη-RFP (C and D) or PKCθ-RFP (E and F). (C and E) Representative time-lapse montages, with the time of UV irradiation indicated by yellow text. Yellow circles denote the irradiated region. All probes were imaged in TIRF. (D and F) Quantification of MyoRLC clearance and nPKC recruitment at the irradiated region (n = 10 cells for each sample). Analysis was performed as in Fig. 3 , with purple lines indicating UV irradiation. Error bars denote SEM. (All scale bars: 5 μm.)

To determine whether nPKC localization was consistent with a role in NMII remodeling, we performed photoactivation experiments using T cells expressing GFP-labeled MyoRLC together with either RFP-labeled PKCθ or RFP-labeled PKCη. Consistent with prior work (5), TCR photoactivation induced the robust accumulation of both PKCη and PKCθ in the irradiated region ( Fig. 6 CF and Movies S6 and S7). PKCη and PKCθ recruitment was markedly anticorrelated with NMII at all time points (Fig. S4A), consistent with the idea that the nPKCs suppress NMII clustering. We also examined the relationship between PKC activity and NMII using a fluorescently labeled form of Marcksl1, a membrane-associated protein that dissociates when phosphorylated by PKCs (5, 23). As expected, photoactivation of T cells expressing GFP-labeled Marcksl1 together with RFP-labeled MyoRLC induced the clearance of both constructs from the irradiated region (Fig. S7 A and B). Depletion of Marcksl1 preceded loss of NMII by 𢏈 s (Fig. S7C). Thus, TCR-induced PKC activation occurred at the right time and place to mediate NMII remodeling.

PKCs are also known to regulate the actin cytoskeleton (24). Thus, it remained possible that the NMII dynamics that we observed were secondary to cortical actin remodeling. To investigate this hypothesis, we imaged T cells expressing GFP-labeled MyoRLC together with RFP-labeled Lifeact, which binds specifically to filamentous (F)-actin (25). TIRF microscopy revealed a splotchy distribution of F-actin beneath the plasma membrane that shifted during cycles of cellular expansion and contraction (Fig. S8A). Although this F-actin partially colocalized with MyoRLC, the formation of NMII clusters was not associated with F-actin enrichment in the same zones. Interestingly, photoactivation of the TCR induced the depletion of F-actin from the irradiated region. This depletion response, however, occurred 24.9 ± 10.0 s after loss of NMII (Fig. S8 B and C), indicating that NMII remodeling is unlikely to be driven by actin in this context.

We also examined the relationship between NMII and peripheral microtubules by imaging T cells expressing MyoRLC-RFP and GFP-tubulin. TIRF microscopy revealed that microtubules close to the plasma membrane were highly dynamic, changing both their length and orientation during polarization responses. There was no clear correlation, however, between these dynamics and NMII remodeling (Fig. S8D). Furthermore, NMII clustering at the rear of the cell was unaffected by depolymerization of microtubules with nocodazole or stabilization with taxol (Fig. S8 E and F). Hence, NMII reorganization occurs independently of microtubules.

Rho-kinase Is Required for NMII Clustering.

Rho kinase (also called ROCK) activates NMII by phosphorylating MyoRLC at Thr18 and Ser19 (12, 26). To test whether ROCK regulates NMII localization in our system, we treated T cells expressing GFP-labeled MyoRLC with Y27632, a ROCK inhibitor. Y27632 induced the dispersion of cortical NMII clusters in less than a minute, similar to the effects of PMA ( Fig. 7 A and B and Movie S8). This dispersion was not reversed by G�, indicating that ROCK is required to stabilize NMII at the plasma membrane, even in the absence of PKC activity ( Fig. 7 A and B ). We also examined whether the ROCK phosphorylation sites within MyoRLC, Thr18 and Ser19, were required for NMII remodeling in photoactivation experiments. Mutation of both residues to Ala markedly reduced MyoRLC clustering at the membrane ( Fig. 7C ), and photoactivation of the TCR did not enhance this clustering or induce asymmetry in the NMII distribution ( Fig. 7 C and D ). Together, these data indicated that ROCK-mediated phosphorylation drives NMII clustering during polarization responses. Consistent with this interpretation, we found that Y27632 delayed centrosome reorientation and also reduced maximum centrosome speed ( Fig. 7 E and F ). We conclude that ROCK and the nPKCs establish polarizing NMII asymmetry in T cells through opposing phosphoregulation of MyoRLC.

ROCK is required for NMII clustering behind the centrosome. (A and B) T cells (5C.C7) expressing MyoRLC-RFP were imaged in TIRF and treated with 50 μM Y27632 and 500 nM G� as indicated during time-lapse acquisition. (A) Representative time-lapse montages, with addition of reagents indicated by the red line. (B) Quantification of MyoRLC clustering as described in Fig. 5 (n = 8 cells per curve). (C and D) Photoactivation experiments were performed using 5C.C7 T cells expressing either wild-type MyoRLC-GFP or MyoRLC(T18AS19A)-GFP. (C) Representative time-lapse montages, with the time of UV irradiation indicated by yellow text. Yellow circles denote the irradiated region. (D) MyoRLC asymmetry was quantified as described in Fig. 3 (n ≥ 10 cells per sample). (E and F) Centrosome polarization in the presence of 50 μM Y27632 or vehicle control was assessed as described in Fig. 2 . (All scale bars: 5 μm.) Purple lines in graphs denote UV irradiation, and error bars indicate SEM. P values were calculated using Student t test. ***P < 0.001.

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What makes a lymphocyte a B cell?

B-cell development in mice 24 and humans 25 has been extensively studied, and the functional rearrangement of the Ig loci is a sine qua non. This occurs via an error-prone process involving the combinatorial rearrangement of the V, D, and J gene segments in the H chain locus and the V and J gene segments in the L chain loci. 26 Susumu Tonegawa was awarded the Nobel Prize in Physiology or Medicine in 1987 for this discovery. In mice and humans, this occurs primarily in fetal liver and adult marrow, culminating in the development of a diverse repertoire of functional VDJH and VJL rearrangements encoding the B-cell receptor (BCR). However, in other species (eg, chickens and rabbits) the development of the preimmune Ig repertoire occurs primarily in GALT, and diversification of the repertoire uses the mechanism of gene conversion. 27,28 The discovery of the recombination activating genes 1/2 (RAG-1/2) in the late 1980s by David Baltimore and colleagues provided the mechanistic explanation for the initial steps of DNA strand breakage in both Ig and T-cell receptor rearrangement (Schatz et al 29 and Oettinger et al 30 ).

Early B-cell development is characterized by the ordered rearrangement of Ig H and L chain loci, and Ig proteins themselves play an active role in regulating B-cell development. 31 Pivotal to understanding how early B-cell development is regulated was the discovery of surrogate L chains (SLCs). Originally identified in murine B-lineage cells, 32 the SLC is a heterodimer consisting of 2 distinct proteins originally designated λ5 and VpreB. These 2 proteins pair with the μ H chain to form the so-called pre-BCR in murine and human pre-B cells. 33 Pre-B cells arise from progenitor (pro-B) cells that express neither the pre-BCR or surface Ig (Figure 1). Whether pre-BCR interactions with ligand(s) can serve as a proliferative stimulus and thereby expand pre-B cells with functional μ H chain rearrangements remains unknown. Although potential pre-BCR ligands have been described, 34,35 the recent crystal structure solution of a soluble form of the human pre-BCR suggests that ligand-independent oligomerization is a likely mechanism of pre-BCR–mediated signaling. 36 Subsequently, BCR expression is requisite for B-cell development and survival in the periphery. 37

Antigen-induced B-cell activation and differentiation in secondary lymphoid tissues are mediated by dynamic changes in gene expression that give rise to the germinal center (GC) reaction (see section on B-cell maturation). The GC reaction is characterized by clonal expansion, class switch recombination (CSR) at the IgH locus, somatic hypermutation (SHM) of VH genes, and selection for increased affinity of a BCR for its unique antigenic epitope through affinity maturation. CSR, also known as isotype-switching, was first demonstrated in the chicken. 38 A decade after the discovery of RAG1/2, Honjo and his colleagues (Muramatsu et al 39 ) demonstrated that CSR and SHM are mediated by an enzyme designated activation-induced cytidine deaminase (AID). Expectedly, B-cell AID expression is induced in GCs where CSR and SHM occur. There are 2 theories on how AID functions to promote antibody diversification. One suggests that AID carries out an “RNA editing” function—not being the source of hypermutator activity, per se, but cooperating with another protein to mediate SHM. 40 A more prevailing view posits that AID participates more directly to effect mutation of IgH genes at the DNA level. 41 Unfortunately, generating a diverse protective Ig repertoire can be deleterious because AID can collaborate with other enzymes to generate chromosomal translocations involving c-myc and the IgH locus in some B-cell lymphomas. 42

Lymphocyte development requires the concerted action of a network of cytokines and transcription factors that positively and negatively regulate gene expression. Marrow stromal cell–derived interleukin-7 (IL-7) is a nonredundant cytokine for murine B-cell development that promotes V to DJ rearrangement and transmits survival/proliferation signals. 43 FLT3-ligand and TSLP play important roles in fetal B-cell development. 24 The cytokine(s) that regulate human B-cell development are not as well understood. 25 However, the presence of normal numbers of circulating B cells in primary immune deficiency patients with mutations in genes encoding the IL-7R argues that B-cell development at this stage of life does not require IL-7R signaling. 44 An informative experiment of nature would be a patient with a null mutation in the IL-7 gene, but no such patient has yet been described. The cytokine (or cytokines) that promote marrow B-cell development at all stages of human life remains unknown.

At least 10 distinct transcription factors regulate the early stages of B-cell development, with E2A, EBF, and Pax5 being particularly important in promoting B-lineage commitment and differentiation. 45 Pax5, originally characterized by its capacity to bind to promoter sequences in Ig loci, may be the most multifunctional transcription factor for B cells. 46 Pax5-deficient mice have an arrest in B-cell development at the transition from DJ to VDJ rearrangement. An important revelation came from the discovery that Pax5 can activate genes necessary for B-cell development and repress genes that play critical roles in development of non–B-lineage cells. Thus, Pax5-deficient pro-B cells harbor the capacity to adapt non–B-lineage fates and develop into other hematopoietic lineages. 47 Pax5 also regulates expression of at least 170 genes, a significant number of them important for B-cell signaling, adhesion, and migration of mature B cells. 46 Conditional Pax5 deletion in mature murine B cells can result in dedifferentiation to an uncommitted hematopoietic progenitor and subsequent differentiation into T-lineage cells under certain conditions. 48 It is remarkable that eliminating the function of this single transcription factor can lead to such a profound change in developmental fate. One obvious question is the whether such dedifferentiation occurs in normal mice (or healthy humans)? Alterations in the Pax5 locus may also have dire consequences mice lacking Pax5 frequently develop high-grade lymphomas. 48 Moreover, up to 30% of newly diagnosed cases of childhood B-lineage ALL harbor somatic PAX5 mutations, representing primarily monoallelic deletions. 49 This provides a fresh perspective on the well-known maturation arrest in early B-cell development that characterizes essentially all childhood ALL cases.


Membrane Association of Tiam1 Is Required for Membrane Ruffling

We have shown previously that full-length Tiam1 (FL1591) or a large COOH-terminal fragment of the Tiam1 protein (C1199) causes Rac1-dependent induction of membrane ruffling in NIH3T3 fibroblast cells (Michiels et al., 1995). Established NIH3T3 cell lines expressing either of these proteins are flat and epithelial-like and contain many membrane ruffles (Fig. 1,B), a phenotype that is also induced by transfection or microinjection of constitutively activated (V12)Rac1 (Ridley et al., 1992 Michiels et al., 1995 Van Leeuwen et al., 1995). In contrast, a smaller COOH-terminal part of the Tiam1 protein (C682), which contains only the DH domain and the adjacent PH domain, did not induce this phenotype (Fig. 1,C). As shown by confocal laser scanning immunofluorescence microscopy, the truncated large C1199 Tiam1 protein was present in the cytoplasm and colocalized with F-actin in membrane ruffles (Fig. 1,B). In contrast, the short C682 Tiam1 protein seemed to be restricted to the cytoplasm (Fig. 1,C). Western blot analyses (see Fig. 4, lanes 1–3) indicated that both proteins were intact and equally expressed. This suggested that the difference in phenotypes induced by these truncated proteins is probably caused by a different intracellular localization, and not by differences in stability. Immunoelectron microscopy (immuno-EM) indeed confirmed that the C1199 Tiam1 protein is present in the cytoplasm as well as at the cell membrane and particularly in the membrane ruffles, whereas the C682 Tiam1 protein is almost exclusively located in the cytoplasm (Fig. 2). We hypothesized, therefore, that membrane localization of Tiam1 is required for morphological transformation of NIH3T3 cells, including the formation of membrane ruffles.

The NH2-terminal PH Domain Is Essential for Membrane Localization of Tiam1

To investigate which of the conserved domains in Tiam1 determines the intracellular localization, small deletions were made in each of these domains within the C1199 construct (see Fig. 3 and Materials and Methods). These mutant proteins were transiently expressed in COS-7 cells to analyze their intracellular localization and ability to induce membrane ruffling. Transfection of these mutant Tiam1 constructs in NIH3T3 cells resulted in the same phenotypic changes (not shown). Although Tiam1 is endogenously expressed in COS-7 cells and NIH3T3 cells, the levels are too low to be visualized by immunocytochemistry or Western blotting. If phenotypic changes were induced by the transfection of Tiam1 mutants, they were found in >80% of the transfected cells.

As shown in Fig. 3,B, the C1199 Tiam1 protein carrying a small internal deletion in the conserved COOH-terminal part of the DH-adjacent PH domain (C1199-ΔPHc) was still able to induce membrane ruffling. This was an unexpected finding since deletions in the DH-adjacent PH domain of other GDS proteins interfered with their proper functioning (Hart et al., 1994 Whitehead et al., 1995b), and mutations in this region of the PH domain of β-Ark abolished interactions with both Gβγ proteins and phospholipids (Touhara et al., 1995). In contrast, a C1199 Tiam1 protein with a deletion in the DH domain (C1199ΔDH) did not induce membrane ruffling (Fig. 3,C), corroborating a previously proposed catalytic function for the DH domain (Hart et al., 1994). Deletion of most of the DHR region (C1199-ΔDHR) did not affect ruffling (Fig. 3,D), indicating that the DHR domain is not required for the induction of membrane ruffles. However, a small deletion in the NH2-terminal PH domain (C1199-ΔPHn), comparable to the deletion made in the COOH-terminal PH domain, completely abolished membrane ruffling (Fig. 3,E). Western blot analyses showed that all mutant proteins were of the predicted size and expressed at similar levels (see Fig. 4, lanes 4–9), although somewhat less than the C1199 Tiam1 protein. This excludes the possibility that these results were due to major differences in protein stability. We conclude that both the catalytic DH domain and the NH2-terminal PH domain are essential for Tiam1-induced membrane ruffling.

Immuno-EM analyses of transfected COS cells showed that all mutant proteins were equally present in the cytoplasm and at the plasma membrane (Fig. 5), except for mutant C1199 Tiam1, which contains a deletion in the NH2terminal PH domain (C1199-ΔPHn). This protein was almost exclusively localized in the cytoplasm (Fig. 5,D), similar to the C682 Tiam1 protein (Fig. 2,B). The C1199-ΔDH Tiam1 protein, which carries a deletion in the catalytic DH domain and does not induce membrane ruffling, was still capable of associating with the plasma membrane (Fig. 5 B). This clearly shows that membrane localization of Tiam1 is not a consequence of the induction of membrane ruffling. Therefore, it can be concluded that the NH2-terminal PH domain is essential for the membrane localization of C1199 Tiam1 and that this membrane localization is required for the formation of membrane ruffles.

The NH2-terminal PH Domain of Tiam1 Can Be Functionally Replaced by the c-Src Membrane Localization Domain, but Not by Other PH Domains

To determine whether membrane localization is the only function of the NH2-terminal PH domain, we fused the NH2terminal 20 amino acids of c-Src, containing the myristoylation site and a basic region, in front of a C580 Tiam1 protein, resulting in M S -C580 Tiam1 (see Materials and Methods). C580 Tiam1 encompasses the COOH-terminal 580 amino acids and encodes the DH domain and adjacent PH domain (Fig. 6,A). Similar to the C682 Tiam1 protein (see Fig. 2,B), the C580 Tiam1 protein did not induce membrane ruffling and was not localized at the plasma membrane (Figs. 6,A and 7,A). In contrast, the M S -C580 Tiam1 protein induced ruffling in both NIH3T3 cells and COS-7 cells (Fig. 6,B), albeit less extensively than C1199 Tiam1 (Fig. 3,A). No differences were observed in the expression levels of these proteins (Fig. 4, lanes 10–12). Immuno-EM showed that M S -C580 Tiam1 was able to associate with the plasma membrane (Fig. 7,B), although this protein seemed to be more interiorly located than the mutant C1199 Tiam1 proteins (compare Figs. 5 and 7,B). Furthermore, part of the expressed M S -C580 Tiam1 protein was present around vesicle-like structures in the cytoplasm, similar to other M S -containing Tiam1 fusion proteins (see for example Fig. 6 E, inset). A fusion protein containing the NH2-terminal Src domain in front of the C1199-ΔPHn Tiam1 protein was also able to localize at the plasma membrane and to cause ruffling (data not shown). The Src membrane localization domain thus enables C580 Tiam1 and C1199-ΔPHn to induce membrane ruffling by tethering these proteins to the plasma membrane.

To test whether the NH2-terminal PH domain of Tiam1 is sufficient for membrane localization, we fused a region containing this PH domain to the C580 Tiam1 protein (see Materials and Methods). The resulting protein, however, did not induce membrane ruffling and was not associated with the plasma membrane (Michiels, F., and J.C. Stam, unpublished results). This might argue that additional sequences are required to ensure membrane localization. Alternatively, the juxtaposition of the PH domain to the DH domain might interfere with the proper folding of these domains. A construct expressing solely the region containing the NH2-terminal PH domain did not function as a dominant-negative Tiam1 mutant. However, C1199-ΔDH, which contains a deletion in the catalytic DH domain, also does not interfere with the induction of membrane ruffling by Tiam1. The reason for this is not clear at the moment.

To analyze the specificity of the NH2-terminal PH domain of Tiam1, a region containing this PH domain was exchanged for the corresponding regions containing the PH domain of DbL, the PH domain of β-ARK, or the COOHterminal PH domain of Tiam1 (see Materials and Methods). However, none of these mutant Tiam1 proteins localized at the plasma membrane and induced membrane ruffling (data not shown). This means either that additional sequences are required for correct functioning of these PH domains or that the primary function of the NH2terminal PH domain, namely to localize Tiam1 through specific interactions with components of the plasma membrane, cannot be substituted for by other PH domains.

The NH2-terminal PH Domain Is Required for the Induction of Membrane Ruffles by the Full-Length Tiam1 Protein

The full-length Tiam1 protein (FL1591) carries a consensus myristoylation sequence at the NH2 terminus. By labeling with [ 3 H]myristate, we have confirmed that the NH2 terminus of Tiam1 is myristoylated (not shown). To test whether the myristoylation is sufficient for the membrane association of full-length Tiam1, a small deletion was also made in the NH2-terminal PH domain of full-length FL1591 Tiam1. However, similar to C1199-ΔPHn, mutant fulllength Tiam1 (FL1591-ΔPHn) was unable to induce membrane ruffling in NIH3T3 cells or COS cells (Fig. 6, C and D), and immuno-EM showed that the protein was not localized at the plasma membrane (data not shown). Apparently, the NH2-terminal myristoylation of full-length Tiam1 alone is not sufficient for membrane localization. To further substantiate this, the NH2-terminal c-Src sequences were fused in front of the FL1591-ΔPHn1 Tiam1 protein. This region of Src contains a basic region that is required for optimal membrane translocation in addition to the myristoylation sequence, whereas other Src-like tyrosine kinases contain a palmitoylated cysteine in this region (Superti-Furga and Courtneidge, 1995). Both additional sequences are absent in Tiam1. Cells expressing the M S -FL1591-ΔPHn1 Tiam1 protein again showed membrane ruffling, although less extensive than cells expressing FL1591 Tiam1 (Fig. 6, C–E). Again, no main differences in expression levels were observed between these proteins (data not shown). Immuno-EM confirmed that the M S -FL1591-ΔPHn1 Tiam1 protein was present at and close to the plasma membrane, as found for M S -C580 Tiam1 (data not shown). Apparently, the accessory basic region of c-Src also functions to properly localize M S -FL1591ΔPHn1 Tiam1. Similar to M S -C580, the expression of M S FL1591-ΔPHn Tiam1 led to the formation of small cytoplasmic vesicle-like structures, and the protein was also located around these structures (Fig. 6 E). These observations indicate that the NH2-terminal PH domain also serves as an essential membrane tag, or perhaps membrane anchor, for full-length Tiam1.

Controlled Cellular Localization of Tiam1

PH domains have been identified in other signaling molecules as protein–protein and/or protein–phospholipid interaction motifs that are required for the controlled targeting of these proteins to the plasma membrane (Lemmon et al., 1996). In tissue sections of skin and certain carcinomas, endogenous Tiam1 is predominantly present in the cytoplasm. Also in some cell lines, where we can envision endogenous Tiam1 by Western blotting, including T-lymphoma cells and neuronal cells, it is mainly localized in the cytoplasmic fraction (data not shown), suggesting that Tiam1 may translocate to the plasma membrane after receptor stimulation. So far, however, we have not been able to identify a receptor-mediated signaling pathway involving Tiam1 activation. To investigate whether membrane translocation of exogenous Tiam1 could be visualized in NIH3T3 cells, we analyzed whether serum could affect the localization and capacity of Tiam1 to induce membrane ruffling. As shown in Fig. 8,A, NIH3T3 cells transiently expressing the C1199 Tiam1 protein showed no membrane ruffling after serum starvation for 24 h. Almost no Tiam1 protein was present at the plasma membrane, and F-actin was mostly concentrated in lamellipodia in the Tiam1-expressing cells. Since these optical sections were taken at the basal site of the cells to illustrate the lamellipodia, stress fibers are also visible. Note that after serum starvation for 24 h, NIH3T3 cells still contain some stress fibers, in contrast to Swiss 3T3 cells. Immuno-EM confirmed that most of the cells contained significantly less Tiam1 at the plasma membrane after serum starvation (Fig. 8, D and F). The residual membrane-associated Tiam1 might be sufficient for the presence of lamellipodia under these conditions. Addition of serum induced membrane localization and subsequent ruffling of C1199 Tiam1-expressing cells after 2 h (Fig. 8, B and E), at which time point seruminduced stress fibers have already decreased. A quantification of these results is presented in Fig. 8,F. Similar results were obtained with FL1591 Tiam1, C1199-ΔPHc, and C1199ΔDHR Tiam1 (data not shown). In contrast, after 24 h of serum starvation the M S -C580 Tiam1 protein remained at the plasma membrane and still induced membrane ruffling (Fig. 8 C). This suggests that the serum-induced membrane translocation of Tiam1 is mediated by the NH2-terminal PH domain.

Unexpectedly, neither PDGF nor insulin could substitute for serum in the induction of membrane ruffling (data not shown). Both growth factors induce lamellipodia formation in NIH3T3 cells within 5 min, which can easily be discriminated from the Tiam1-induced ruffling. This might indicate that Tiam1 is not activated by PDGF or insulin.

Membrane-associated Tiam1 Activates JNK

To investigate whether membrane association of Tiam1 is also required for the induction of other Rac-mediated signaling pathways besides membrane ruffling, the activity of JNK was determined after cotransfection with different Tiam1 mutants (see Materials and Methods). Both in COS-7 cells and NIH3T3 cells, cotransfection of fulllength Tiam1 led to an almost sevenfold stimulation of JNK activity, similar to constitutively activated (V12)Rac1 (Fig. 9,A). Cotransfection of tagged mitogen-activated protein kinase (MAPK) with full-length or mutant Tiam1 or (V12)Rac did not result in activation of MAPK (not shown). The stimulation of JNK by Tiam1 was dependent on the activation of Rac, since cotransfection of dominantnegative (N17)Rac1 partly blocked the activation of JNK by Tiam1 (Fig. 9,A). Higher amounts of (N17)Rac interfered with the expression of JNK. Furthermore, a deletion in the catalytic DH domain of Tiam1 prevented JNK activation (not shown). Cotransfection of dominant-negative (N17)Cdc42 did not interfere with the activation of JNK by Tiam1 (data not shown). Transfection of C1199 Tiam1 stimulated JNK activity to a similar extent as FL1591 (Fig. 9,B). However, shorter COOH-terminal Tiam1 fragments such as C682 and C580 Tiam1, which did not localize to the plasma membrane, were not able to activate JNK above background levels. Also, mutant C1199-ΔPHn Tiam1, which did not localize to the plasma membrane because of the deletion in the NH2-terminal PH domain, was unable to activate JNK (Fig. 9,B). M S -C580 Tiam1 caused no stimulation of JNK activity (Fig. 9,B), and neither did M S -C1199-ΔPHn or M S -FL1591-ΔPHn Tiam1 proteins (data not shown). These proteins, however, induced membrane ruffling and were present at the plasma membrane (Fig. 6). A possible explanation for this somewhat unexpected result is that activation of endogenous Rac by M S -containing Tiam1 proteins is sufficient for induction of membrane ruffling but not for stimulation of JNK. Alternatively, the lack of induction of JNK activity is caused by the different ultrastructural localization of the M S -C580 Tiam1 protein (compare Figs. 5 and 7) or may reflect the need for an intact NH2-terminal PH domain. Interestingly, similar results were obtained with membrane-targeted p110 phosphoinositide-3-kinase which was able to stimulate membrane ruffling but not gene transcription from the Jun-inducible Fos promoter (Reif et al., 1996). Whatever the mechanism, these results indicate that the localization of Tiam1 at the plasma membrane is also required for Rac-mediated stimulation of JNK activity.


The microbe-specific molecules that are recognized by a given PRR are called pathogen-associated molecular patterns (PAMPs) and include bacterial carbohydrates (such as lipopolysaccharide or LPS, mannose), nucleic acids (such as bacterial or viral DNA or RNA), bacterial peptides (flagellin, microtubule elongation factors), peptidoglycans and lipoteichoic acids (from Gram-positive bacteria), N-formylmethionine, lipoproteins and fungal glucans and chitin.

Endogenous stress signals are called damage-associated molecular patterns (DAMPs) and include uric acid and extracellular ATP, among many other compounds. [2]

There are several subgroups of PRRs. They are classified according to their ligand specificity, function, localization and/or evolutionary relationships. Based on their localization, PRRs may be divided into membrane-bound PRRs and cytoplasmic PRRs.

Membrane-bound PRRs Edit

Receptor kinases Edit

PRRs were first discovered in plants. [6] Since that time many plant PRRs have been predicted by genomic analysis (370 in rice 47 in Arabidopsis). Unlike animal PRRs, which associated with intracellular kinases via adaptor proteins (see non-RD kinases below), plant PRRs are composed of an extracellular domain, transmembrane domain, juxtamembrane domain and intracellular kinase domain as part of a single protein.

Toll-like receptors (TLR) Edit

Recognition of extracellular or endosomal pathogen-associated molecular patterns is mediated by transmembrane proteins known as toll-like receptors (TLRs). [7] TLRs share a typical structural motif, the Leucine rich repeats (LRR), which give them their specific appearance and are also responsible for TLR functionality. [8] Toll-like receptors were first discovered in Drosophila and trigger the synthesis and secretion of cytokines and activation of other host defense programs that are necessary for both innate or adaptive immune responses. 10 functional members of the TLR family have been described in humans so far. [5] Studies have been conducted on TLR11 as well, and it has been shown that it recognizes flagellin and profilin-like proteins in mice. [9] Nonetheless, TLR11 is only a pseudogene in humans without direct function or functional protein expression. Each of the TLR has been shown to interact with a specific PAMP. [5] [10] [11]

The TLR signaling Edit

TLRs tend to dimerize, TLR4 forms homodimers, and TLR6 can dimerize with either TLR1 or TLR2. [10] Interaction of TLRs with their specific PAMP is mediated through either MyD88- dependent pathway and triggers the signaling through NF-κB and the MAP kinase pathway and therefore the secretion of pro-inflammatory cytokines and co-stimulatory molecules or TRIF - dependent signaling pathway. [2] [5] [10] MyD88 - dependent pathway is induced by various PAMPs stimulating the TLRs on macrophages and dendritic cells. MyD88 attracts the IRAK4 molecule, IRAK4 recruits IRAK1 and IRAK2 to form a signaling complex. The signaling complex reacts with TRAF6 which leads to TAK1 activation and consequently the induction of inflammatory cytokines. The TRIF-dependent pathway is induced by macrophages and DCs after TLR3 and TLR4 stimulation. [2] Molecules released following TLR activation signal to other cells of the immune system making TLRs key elements of innate immunity and adaptive immunity. [2] [12] [13]

C-type lectin receptors (CLR) Edit

Many different cells of the innate immune system express a myriad of CLRs which shape innate immunity by virtue of their pattern recognition ability. [14] Even though, most classes of human pathogens are covered by CLRs, CLRs are a major receptor for recognition of fungi: [15] [16] nonetheless, other PAMPs have been identified in studies as targets of CLRs as well e.g. mannose is the recognition motif for many viruses, fungi and mycobacteria similarly fucose presents the same for certain bacteria and helminths and glucans are present on mycobacteria and fungi. In addition, many of acquired nonself surfaces e.g. carcinoembryonic/oncofetal type neoantigens carrying "internal danger source"/"self turned nonself" type pathogen pattern are also identified and destroyed (e.g. by complement fixation or other cytotoxic attacks) or sequestered (phagocytosed or ensheathed) by the immune system by virtue of the CLRs. The name lectin is a bit misleading because the family includes proteins with at least one C-type lectin domain (CTLD) which is a specific type of carbohydrate recognition domain. CTLD is a ligand binding motif found in more than 1000 known proteins (more than 100 in humans) and the ligands are often not sugars. [17] If and when the ligand is sugar they need Ca2+ – hence the name "C-type", but many of them don't even have a known sugar ligand thus despite carrying a lectin type fold structure, some of them are technically not "lectin" in function.

CLR signaling Edit

There are several types of signaling involved in CLRs induced immune response, major connection has been identified between TLR and CLR signaling, therefore we differentiate between TLR-dependent and TLR-independent signaling. DC-SIGN leading to RAF1-MEK-ERK cascade, BDCA2 signaling via ITAM and signaling through ITIM belong among the TLR-dependent signaling. TLR-independent signaling such as Dectin 1, and Dectin 2 - mincle signaling lead to MAP kinase and NFkB activation. [18] [15]

Membrane receptor CLRs have been divided into 17 groups based on structure and phylogenetic origin. [19] Generally there is a large group, which recognizes and binds carbohydrates, so called carbohydrate recognition domains (CRDs) and the previously mentioned CTLDs.

Another potential characterization of the CLRs can be into mannose receptors and asialoglycoprotein receptors. [18]

Group I CLRs: The mannose receptors Edit

The mannose receptor (MR) [20] is a PRR primarily present on the surface of macrophages and dendritic cells. It belongs into the calcium-dependent multiple CRD group. [15] The MR belongs to the multilectin receptor protein group and, like the TLRs, provides a link between innate and adaptive immunity. [21] [22] It recognizes and binds to repeated mannose units on the surfaces of infectious agents and its activation triggers endocytosis and phagocytosis of the microbe via the complement system. Specifically, mannose binding triggers recruitment of MBL-associated serine proteases (MASPs). The serine proteases activate themselves in a cascade, amplifying the immune response: MBL interacts with C4, binding the C4b subunit and releasing C4a into the bloodstream similarly, binding of C2 causes release of C2b. Together, MBL, C4b and C2a are known as the C3 convertase. C3 is cleaved into its a and b subunits, and C3b binds the convertase. These together are called the C5 convertase. Similarly again, C5b is bound and C5a is released. C5b recruits C6, C7, C8 and multiple C9s. C5, C6, C7, C8 and C9 form the membrane attack complex (MAC).

Group II CLRs: asialoglycoprotein receptor family Edit

This is another large superfamily of CLRs that includes

  1. The classic asialoglycoprotein receptor macrophage galactose-type lectin (MGL) (CLEC4L) (CLEC4K) (CLEC5A)
  2. DC‑associated C‑type lectin 1 (Dectin1) subfamily which includes
      /CLEC7A /CLEC9A
  3. Myeloid C‑type lectin‑like receptor (MICL) (CLEC12A)
  4. CLEC2 (also called CLEC1B)- the platelet activation receptor for podoplanin on lymphatic endothelial cells and invading front of some carcinomas.
    1. /CLEC4A /CLEC6A
    2. Blood DC antigen 2 (BDCA2) ( CLEC4C) i.e. macrophage‑inducible C‑type lectin (CLEC4E)

    The nomenclature (mannose versus asialoglycoprotein) is a bit misleading as these the asialoglycoprotein receptors are not necessarily galactose (one of the commonest outer residues of asialo-glycoprotein) specific receptors and even many of this family members can also bind to mannose after which the other group is named.

    Cytoplasmic PRRs Edit

    NOD-like receptors (NLR) Edit

    For more details, see NOD-like receptor.

    The NOD-like receptors (NLRs) are cytoplasmic proteins, which recognize bacterial peptidoglycans and mount proinflammatory and antimicrobial immune response. [23] Approximately 20 of these proteins have been found in the mammalian genome and include nucleotide-binding oligomerization domain (NODs), which binds nucleoside triphosphate. Among other proteins the most important are: the MHC Class II transactivator (CIITA), IPAF, BIRC1 etc. [24]

    NLR signaling Edit

    Some of these proteins recognize endogenous or microbial molecules or stress responses and form oligomers that, in animals, activate inflammatory caspases (e.g. caspase 1) causing cleavage and activation of important inflammatory cytokines such as IL-1, and/or activate the NF-κB signaling pathway to induce production of inflammatory molecules.

    The NLR family is known under several different names, including the CATERPILLER (or CLR) or NOD-LRR family. [24] [25] The most significant members of the NLRs are NOD1 and NOD2. They sense the conserved microbial peptidoglycans in the cytoplasm of the cell and therefore represent another level of immune response after membrane-bound receptors such as TLRs and CLRs. [23] This family of proteins is greatly expanded in plants, and constitutes a core component of plant immune systems. [26]

    NODs Edit
    NLRPs Edit
    Other NLRs Edit

    RIG-I-like receptors (RLR) Edit

    Three RLR helicases have so far been described: RIG-I and MDA5 (recognizing 5'triphosphate-RNA and dsRNA, respectively), which activate antiviral signaling, and LGP2, which appears to act as a dominant-negative inhibitor. RLRs initiate the release of inflammatory cytokines and type I interferon (IFN I). [2]

    RLR signaling Edit

    RLRs, are RNA helicases, which have been shown to participate in intracellular recognition of viral double-stranded (ds) and single stranded RNA which recruit factors via twin N-terminal CARD domains to activate antiviral gene programs, which may be exploited in therapy of viral infections. [32] [33] It has been suggested that the main antiviral program induced by RLR is based on ATPase activity. [34] RLRs often interact and create cross-talk with the TLRs in the innate immune response and in regulation of adaptive immune response. [35]

    RIG-I and Mda5-mediated signalling pathway.

    Plants contain a significant number of PRRs that share remarkable structural and functional similarity with drosophila TOLL and mammalian TLRs. The first PRR identified in plants or animals was the Xa21 protein, conferring resistance to the Gram-negative bacterial pathogen Xanthomonas oryzae pv. oryzae. [6] [36] Since that time two other plants PRRs, Arabidopsis FLS2 (flagellin) and EFR (elongation factor Tu receptor)have been isolated. [37] The corresponding PAMPs for FLS2 and EFR have been identified. [37] Upon ligand recognition, the plant PRRs transduce "PAMP-triggered immunity" (PTI). [38] Plant immune systems also encode resistance proteins that resemble NOD-like receptors (see above), that feature NBS and LRR domains and can also carry other conserved interaction domains such as the TIR cytoplasmic domain found in Toll and Interleukin Receptors. [39] The NBS-LRR proteins are required for effector triggered immunity (ETI).

    PRRs commonly associate with or contain members of a monophyletic group of kinases called the interleukin-1 receptor-associated kinase (IRAK) family that include Drosophila Pelle, human IRAKs, rice XA21 and Arabidopsis FLS2. In mammals, PRRs can also associate with members of the receptor-interacting protein (RIP) kinase family, distant relatives to the IRAK family. Some IRAK and RIP family kinases fall into a small functional class of kinases termed non-RD, many of which do not autophosphorylate the activation loop. A survey of the yeast, fly, worm, human, Arabidopsis, and rice kinomes (3,723 kinases) revealed that despite the small number of non-RD kinases in these genomes (9%–29%), 12 of 15 kinases known or predicted to function in PRR signaling fall into the non-RD class. In plants, all PRRs characterized to date belong to the non-RD class. These data indicate that kinases associated with PRRs can largely be predicted by the lack of a single conserved residue and reveal new potential plant PRR subfamilies. [40] [41]

    A number of PRRs do not remain associated with the cell that produces them. Complement receptors, collectins, ficolins, pentraxins such as serum amyloid and C-reactive protein, lipid transferases, peptidoglycan recognition proteins (PGRPs) [42] and the LRR, XA21D [43] are all secreted proteins. One very important collectin is mannan-binding lectin (MBL), a major PRR of the innate immune system that binds to a wide range of bacteria, viruses, fungi and protozoa. MBL predominantly recognizes certain sugar groups on the surface of microorganisms but also binds phospholipids, nucleic acids and non-glycosylated proteins. [44] Once bound to the ligands MBL and Ficolin oligomers recruit MASP1 and MASP2 and initiate the lectin pathway of complement activation which is somewhat similar to the classical complement pathway.

    Research groups have recently conducted extensive research into the involvement and potential use of patient's immune system in the therapy of various diseases, the so-called immunotherapy, including monoclonal antibodies, non-specific immunotherapies, oncolytic virus therapy, T-cell therapy and cancer vaccines. [45] NOD2 has been associated through a loss- and gain- of function with development of Crohn's disease and early-onset sarcoidosis. [23] [46] Mutations in NOD2 in cooperation with environmental factors lead to development of chronic inflammation in the intestine. [23] [47] Therefore, it has been suggested to treat the disease by inhibiting the small molecules, which are able to modulate the NOD2 signaling, particularly RIP2. Two therapeutics have been approved by FDA so far inhibiting the phosphorylation on RIP2, which is necessary for proper NOD2 functioning, gefitinib and erlotinib. [48] [49] Additionally, research has been conducted on GSK583, a highly specific RIP2 inhibitor, which seems highly promising in inhibiting NOD1 and NOD2 signaling and therefore, limiting inflammation caused by NOD1, NOD2 signaling pathways. [50] Another possibility is to remove the sensor for NOD2, which has been proved efficient in murine models in the effort to suppress the symptoms of Crohn's disease. [51] Type II kinase inhibitors, which are highly specific, have shown promising results in blocking the TNF arising from NOD-dependent pathways, which shows a high potential in treatment of inflammation associated tumors. [52]

    Another possible exploitation of PRRs in human medicine is also related to tumor malignancies of the intestines. Helicobacter pylori has been shown by studies to significantly correlate with the development of a gastrointestinal tumors. In a healthy individual Helicobacter pylori infection is targeted by the combination of PRRs, namely TLRs, NLRs, RLRs and CLR DC-SIGN. In case of their malfunction, these receptors have also been connected to carcinogenesis. When the Helicobacter pylori infection is left to progress in the intestine it develops into chronic inflammation, atrophy and eventually dysplasia leading to development of cancer. Since all types of PRRs play a role in the identification and eradication of the infection, their specific agonists mount a strong immune response to cancers and other PRR-related diseases. The inhibition of TLR2 has been shown to significantly correlate with improved state of the patient and suppression of the gastric adenocarcinoma. [53]

    The PRRs are also tightly connected to the proper function of neuronal networks and tissues, especially because of their involvement in the processes of inflammation, which are essential for proper function but may cause irreparable damage if not under control. The TLRs are expressed on most cells of the central nervous system (CNS) and they play a crucial role in sterile inflammation. After an injury, they lead to impairment of axonal growth and slow down or even halt the recovery altogether. Another important structure involved in and potentially exploitable in therapy after injury is the inflammasome. Through its induction of proinflammatory cytokines, IL-1β and IL-18 it has been proposed, that inhibition of inflammasome may also serve as an efficient therapeutic method. [54] The involvement of inflammasome has also been researched in several other diseases including experimental autoimmune encephalomyelitis (EAE), Alzheimer's and Parkinson's diseases and in atherosclerosis connected with type II diabetes in patients. The suggested therapies include degradation of NLRP3 or inhibit the proinflammatory cytokines. [54]

    Concluding Remarks

    Based on the data discussed in this review we propose that the evolutionary conservation of two CPC centromere recruitment arms ensures that sufficient amounts of active Aurora B accumulate in the vicinity of kinetochores. We suggest that it makes the KT-MT error correction system robust, thereby making dividing cells more resilient to conditions that would weaken the mitotic checkpoint or that would increase the chance of acquiring erroneous KT-MT attachments, such as disturbances in the geometry of the mitotic spindle (Ertych et al., 2014). In other words, it may safeguard chromosome segregation fidelity in anomalous situations. In addition, inner centromere localization of Aurora B may still be relevant to control the phosphorylation status of a number of outer kinetochore substrates, but not all of them. This likely also depends on the regulation of the phosphatase that dephosphorylates a particular substrate. If the antagonizing phosphatase is present at the kinetochore in relatively high amounts then even a slight change in the kinase-substrate distance may have a dramatic effect on the phosphorylation status of the substrate. Finally, the realization that the CEN module of the CPC strengthens centromere cohesion and is involved in inner kinetochore assembly opens up the possibility that the �tivity” of the CEN module requires inner centromere localization.


    In this study, we investigated the roles of the N- and C-terminal regions of P4-ATPases with respect to their cellular localization. Importantly, we found that the NT regions of the ATP10 family and the CT regions of the ATP11 family are critical for their cellular localization, suggesting that these cytoplasmic regions include specific membrane-targeting and/or retention signals. Moreover, the N-­terminus of ATP10B and the C-terminus of ATP11B serve as endosomal targeting signals for these enzymes. The N-termini of ATP9B and ATP13A2, a P5-ATPase, are required for their Golgi and late endosomal localization, respectively (Takatsu et al., 2011 Holemans et al., 2015). Moreover, the N-termini of ATP9B and ATP13A2 alone are sufficient for their localization through their interactions with the membranes of specific organelles. However, the N-terminus of ATP10B and the C-terminus of ATP11B were not sufficient for targeting to specific endosomal membranes because the N- and C-termini remained distributed in the cytosol when expressed alone (Supplemental Figure S1). By striking contrast, when the plasma membrane–targeting Lyn11 sequence was fused to ATP10B-NT or ATP11B-CT, the chimeric constructs became localized to specific endosomal compartments (late endosomes or early/recycling endosomes, respectively) but not to the plasma membrane. Therefore, ATP10B-NT and ATP11B-CT likely serve as intracellular trafficking and targeting signals to direct the proteins from the plasma membrane to specific endosomal compartments. Many transmembrane proteins that localize to intracellular compartments, including Lamp-1, TGN46, and mannose-6-phosphate receptor, cycle between the plasma membrane and endosomes (Dell’Angelica et al., 1999 Ishizaki et al., 2008 Nakai et al., 2013), and ATP11A and ATP11C, which localize to the plasma membrane, also cycle between the plasma membrane and endosomes (Steinberg et al., 2013 Takatsu et al., 2017). The cell surface expression of ATP11A and ATP11C is reduced in SNX27- or VPS35-depleted cells (Steinberg et al., 2013). Thus, SNX27 and the retromer complex regulate the recycling of ATP11A and ATP11C from endosomes to the plasma membrane. Taken together, these data suggest that ATP10B and ATP11B traffic between intracellular compartments and the plasma membrane, and they possess an endosomal targeting and/or retention signal within their NT and CT regions, respectively.

    As the NT sequences are diverse among the ATP10 proteins, these regions may play specific roles in individual proteins (Supplemental Figure S2). Consistent with this, the N-terminus is critical for determining the localization of ATP10A, ATP10B, and ATP10D, and the N-terminus of ATP10B is sufficient for the late endosomal targeting of plasma membrane proteins, including Lyn11-EGFP. In addition, ATP10B-NT contains a dileucine-like ExxxLL motif (Supplemental Figure S2), and indeed the two leucine residues are required for the trafficking of Lyn11-EGFP-ATP10B-NT from the plasma membrane to late endosomes (Figure 7), while the Glu residue seems to be dispensable. The P1B-ATPases, ATP7A and ATP7B, also harbor the dileucine-like intracellular trafficking signals in their CT regions (Greenough et al., 2004 Lalioti et al., 2014). It remains to be elucidated whether ATP10B is transported from the plasma membrane to late endosomes or transported from the Golgi complex directly to endosomes during biosynthesis. Nevertheless, when ATP10B is transported to the plasma membrane, the dileucine motif serves as a trafficking signal for targeting to late endosomes (Figure 8). Notably, although D-ATP10B localized to the plasma membrane, Lyn11-EGFP-ATP10D-NT appeared in some punctate structures within the cytoplasm as well as at the plasma membrane. Therefore, the N-terminus of ATP10D might also contain a signal for endocytosis.

    We previously showed that the C-terminal SVRPLL sequence of ATP11C-a serves as a dileucine-like motif when the Ser is phosphorylated by protein kinase C (PKC) α and subsequently down-regulated by endocytosis (Supplemental Figure S3 Takatsu et al., 2017). Moreover, the C-terminal LLxYKH sequence of ATP11C-b is critical for its polarized localization (Takayama et al., 2019). Therefore, the C-termini of ATP11 proteins play a key role in the regulation of their cellular localization and enzymatic activity at the plasma membrane. Indeed, the C-terminus can determine the localization of ATP11A, ATP11B, and ATP11C (Figure 4), and the C-terminus of ATP11B is sufficient to target plasma membrane proteins, including Lyn11-EGFP, to early/recycling endosomes (Figure 6). Although the endocytic signaling residues of ATP11B-CT are unknown, there might be a signal for trafficking and/or targeting to endosomes.

    The C-terminal cytoplasmic region of Drs2p binds to phosphatidylinositol-4-phosphate (PI4P), and this interaction regulates the enzymatic activity of Drs2p. The C-terminus of Drs2p exhibits autoinhibitory activity, and the interaction with PI4P appears to relieve the inhibitory effect (Natarajan et al., 2009 Zhou et al., 2013 Azouaoui et al., 2017 Timcenko et al., 2019). Moreover, the C-terminus of ATP8A2 undergoes phosphorylation that relieves its autoinhibition (Chalat et al., 2017). In addition, the C-terminal GYAFS motif of ATP8A1 binds to the N-domain of ATP8A1 in the E2P state (Hiraizumi et al., 2019). Therefore, the C-terminus seems to be involved in the regulation of the enzymatic activities of these proteins. On the other hand, swapping the N- or C-termini of ATP10 proteins retained their enzymatic activity or substrate specificity (Figure 3), and swapping the C-terminus of ATP11C did not affect its PS/PE-flipping activity (Takatsu et al., 2017). Because there is a great deal of variation among the NT and CT sequences of P4-ATPases (Supplemental Figures S2 and S3), these cytoplasmic regions may play specific regulatory roles with respect to their enzymatic activity and intracellular trafficking.

    Difference Between T Cells and B Cells


    T Cells: T cells are a type of lymphocyte, which develops in the thymus, circulates in the blood and lymph and mediates the immune response against malignant or infected cells in the body by the secretion of lymphokines or by direct contact.

    B Cells: B cells are a type of lymphocyte, which develops in the bone marrow, circulates in the blood and lymph, and upon recognizing a particular pathogen, differentiates into a plasma cell clone, secreting specific antibodies and a memory cell clone, for the subsequent encountering of the same pathogen.


    T Cells: T cells originate in the bone marrow and mature in the thymus.

    B Cells: B cells originate and mature in the bone marrow.


    T Cells: Mature T cells occur inside the lymph nodes.

    B Cells: Mature B cells occur outside the lymph nodes.

    Membrane Receptor

    T Cells: T cells bear TCR receptor.

    B Cells: B cells bear BCR receptor.

    Recognition of Antigens

    T Cells: T cells recognize viral antigens on the outside of the infected cells.

    B Cells: B cells recognize antigens on the surface of the bacteria and viruses.


    T Cells: T cells occur in the parafollicular areas of the cortex of the lymph nodes and the periarteriolar lymphoid sheath of the spleen.

    B Cells: B cells occur in the germinal centers, subcapsular and medullary cords of lymph nodes, spleen, gut, and the respiratory tract.


    T Cells: The T cells have longer lifespans.

    B Cells: The lifespan of the B cells is short.

    Surface Antibodies

    T Cells: The T cells lack surface antigens.

    B Cells: The B cells have surface antigens.


    T Cells: The T cells secrete lymphokines.

    B Cells: The B cells secrete antibodies.

    Type of Immunity

    T Cells: The T cells are involved in the cell-mediated immunity (CMI).

    B Cells: The B cells are involved in the humoral or the antibody-mediated immunity (AMI).

    Proportions in the Blood

    T Cells: The 80% of the blood lymphocytes are T cells.

    B Cells: The 20% of the blood lymphocytes are B cells.


    T Cells: The three types of T cells are helper T cells, cytotoxic T cells, and suppressor T cells.

    B Cells: The two types of B cells are plasma cells and memory cells.

    Movement to the Infected Site

    T Cells: The T cells move to the site of infection.

    B Cells: The B cells do not move to the site of infection.

    Tumor Cells and Transplants

    T Cells: The T cells act against tumor cells and transplants.

    B Cells: The B cells do not act against tumor cells or transplants.

    Inhibitory Effect

    T Cells: The suppressor T cells have an inhibitory effect on the immune system.

    B Cells: The B cells do not have any inhibitory effect on the immune system.

    Defend against

    T Cells: The T cells defend against the pathogens including viruses, protists, and fungi that enter the cells in the body.

    B Cells: The B cells defend against bacteria and viruses in the bloodstream or lymph.


    T cells and B cells are two types of lymphocytes which trigger an immune response against foreign materials in the body. T cells recognize the foreign antigens on the surface of the APSs. The helper T cells stimulate the production of antibodies by plasma cells. The cytotoxic T cells destroy pathogens by inducing the apoptosis. The B cells produce specific antibodies to different pathogens, by recognizing the antigens in the circulation system. The main difference between T cells and B cells is their method of recognizing antigens.

    Watch the video: How T Cells Work (July 2022).


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