Interorganelle Tethering to Endocytic Organelles Determines Directional Cytokine Transport in CD4⁺ T Cells

Transport of TNF-α and IL-2 is directionally distinct

To investigate the transportation of cytokines, we focused on TNF-α and IL-2 as representative examples. We used primary human CD4⁺ T cells stimulated with anti-CD3/anti-CD28 Ab-coated beads. We found that in these T cells, both TNF-α and IL-2 are upregulated upon IS formation of T cells with anti-CD3/anti-CD28 Ab-coated coverslips. TNF-α mRNA level was already enhanced 30 min after IS formation (Supplemental Fig. 1A), whereas the first peak of IL-2 mRNA appeared at 60 min after IS formation (Supplemental Fig. 1B). Quantification of immunostaining shows that the expression of endogenous IL-2 is initiated later than TNF-α and reached a comparable level of TNF-α 2 h after IS formation (Supplemental Fig. 1C).

Next, we verified the localization of endogenous TNF-α and IL-2 in primary human CD4⁺ T cells at various time points upon IS formation. We settled CD4⁺ T cells on glass coverslips coated with anti-CD3/anti-CD28 Abs to obtain a spatially well-defined IS between the T cell and the coverslip. Deconvolution microscopy results show that TNF-α first appeared in a condensed structure in vicinity to the IS, and then TNF-α–containing vesicles (hereafter referred to as TNF-α⁺ vesicles) were transported multidirectionally (Fig. 1A, upper panel). The condensed structure is colocalized with Golgi marker GM130 (Supplemental Fig. 1D). In comparison with TNF-α, endogenous IL-2 first appeared 60 min after activation and is mainly accumulated at the IS (Fig. 1A, lower panel). Further analysis of the distribution of endogenous TNF-α and IL-2 in the same T cells (Fig. 1B) showed that at 60 min after IS formation, ∼50% of the TNF-α⁺ vesicles were at the IS. With time, at 90 min, fewer TNF-α⁺ vesicles (∼38%) stayed at the IS; in other words, more TNF-α⁺ vesicles were transported away from the IS (i.e., multidirectionally). In contrast, IL-2⁺ vesicles were enriched at the IS already at early time points (60 min, 58%), and their number further increased with time (90 min, ∼64%/120 min, ∼61%). In conclusion, simultaneous recordings from the same cell revealed that, at later time points, IL-2 is enriched at the IS, whereas TNF-α is more multidirectionally distributed, which was especially prominent at 90 min. We noticed that at 120 min, the fraction of TNF-α at the IS was slightly enhanced compared with 90 min. This might be due to newly generated TNF-α⁺ vesicles or the release of TNF-α⁺ vesicles away from the IS. Thus, we confirm that transportation of TNF-α and IL-2 is directionally different, namely TNF-α multidirectionally and IL-2 to the IS.

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FIGURE 1.

TNF-α⁺ vesicles are juxtaposed to endocytic organelles. (A and B) TNF-α⁺ vesicles and IL-2⁺ vesicles are distinctively transported after IS formation. CD4⁺ T cells were settled on CD3/CD28 Ab-coated coverslips. Endogenous TNF-α or IL-2 was stained with corresponding mAb conjugated with Alexa Fluor 488 or Alexa Fluor 647. Maximum intensity projections (MIP) of the exemplary cells from three independent experiments are shown in (A). Quantification of distribution of TNF-α and IL-2 in the same T cells is shown in (B). The segment encompassing one fourth of the cell’s diameter close to the coverslip was defined as IS area (@IS). The fraction of IL-2 or TNF-α (percentage of total number) was analyzed from three independent experiments. Mann–Whitney U test was used. (C and D) Subcellular localization of TNF-α in primary human CD4⁺ T cells. Fluorescently tagged TNF-α and indicated markers for each particular intracellular compartment were transiently overexpressed in stimulated CD4⁺ T cells. Quantification of the tethering rate of TNF-α⁺ vesicles with endosomal markers is shown in (D) (from two independent experiments). (E) Colocalization of Rab7a and LAMP1. Anti-CD3/anti-CD28 Ab-coated beads–stimulated primary human CD4⁺ T cells were transiently overexpressed with LAMP1-EGFP and mCherry-Rab7a. One representative cell out of two independent experiments is shown. (F) TNF-α⁺ vesicles are tethered with LAMP3. Stimulated primary human CD4⁺ T cells transiently overexpressing mCherry-TNF-α and LAMP3-EGFP (n = 3 independent experiments). The samples in (A)–(F) were scanned by deconvolution microscopy with a ×63 objective. (G) Endogenous TNF-α and LAMP1 were stained with Alexa Fluor 647–labeled anti-human TNF-α mAb and Alexa Fluor 488–labeled anti-human LAMP1 mAb. The samples were scanned by superresolution SIM with a ×63 objective. One representative cell out of three independent experiments is shown. (H–J) TNF-α–lysosome tethering is increased with time upon stimulation. Bead-stimulated CD4⁺ T cells were settled on anti-CD3/anti-CD28 Ab-coated coverslips and fixed at indicated time points. Exemplary cells are shown in (H). The fractions of TNF-α⁺ vesicles tethered with LAMP1⁺ vesicles and the numbers of TNF-α⁺ vesicles are shown in (I) and (J), respectively. Twenty-four cells from three independent experiments were analyzed. (K). The tethering rate was not affected by overexpression of TNF-α. Results of endogenous and overexpressed TNF-α are from six and five independent experiments, respectively. Scale bars, 5 μm, except in (C) (3 μm). **p < 0.01, ***p < 0.001.

CytVs are tethered with lysosomes/late endosomes

To reveal how this directionally distinct transport of CytVs is regulated, we examined their subcellular localization. Various organelle markers, such as early (Rab5), late (Rab7), recycling endosomes (Rab11), or lysosomes (LAMP1), were cotransfected with fluorescently tagged TNF-α. We found that TNF-α⁺ vesicles were positioned in close vicinity to particular endosomal compartments, especially late endosomes (Rab7a⁺) and lysosomes (LAMP1⁺) (Fig. 1C, 1D). This consistent juxtaposition of the two vesicles suggests that they may be physically tethered.

To distinguish whether Rab7a and LAMP1 compartments colocalize or tether independently with TNF-α⁺ vesicles, we cotransfected mCherry-Rab7a and LAMP1-EGFP in bead-activated CD4⁺ T cells. We observed that a large proportion of Rab7a colocalized with LAMP1 (Fig. 1E, Pearson correlation coefficient = 0.78 ± 0.05, n = 10). To further explore to which organelle type TNF-α⁺ vesicles are juxtaposed, we used LAMP3 (CD63), another marker for lysosomes. We found that the LAMP3⁺ compartment was also juxtaposed to TNF-α⁺ vesicles (Fig. 1F). To investigate whether endogenous TNF-α⁺ vesicles are also juxtaposed to lysosomes, we used superresolution SIM. We observed the vesicles containing endogenous TNF-α to be in close vicinity of lysosomes marked by endogenous LAMP1 (Fig. 1G). Thus, we conclude that TNF-α⁺ vesicles are juxtaposed to late endosomes/lysosomes. Hereafter, we used LAMP1 as the marker for late endosomes/lysosomes.

Next, we analyzed whether this juxtaposition could be changed over time upon IS formation. The immunostaining results show that the percentage of endogenous TNF-α juxtaposed to lysosomes was significantly increased from <40% at 60 min to around 60% at 90 min after IS formation and stayed relatively stable afterward (Fig. 1H, 1I). The total numbers of TNF-α⁺ vesicles remained unchanged during this period of time (Fig. 1J). In addition, we found no difference in the fraction of LAMP1-tethered TNF-α between endogenous and overexpression cases (Fig. 1K), indicating that overexpression of TNF-α does not change the tethering rate. These results suggest that tethering between lysosomes and TNF-α⁺ vesicles may be promoted by IS formation.

Considering that the transport patterns of TNF-α (multidirectional) and IL-2 (IS polarized) are different, we hypothesized that, in contrast to TNF-α⁺ vesicles, IL-2⁺ vesicles will not tether with endocytic organelles. However, we found that the vesicles containing endogenous IL-2 were also juxtaposed to LAMP1⁺ organelles at all time points examined upon the formation of IS with no significant difference (Fig. 2A, 2B). Noticeably, the overall number of IL-2⁺ vesicles was decreased at 150 min compared with earlier time points (Fig. 2C), likely because of their release. To establish whether the two organelles were truly close enough to be physically tethered to one another, we performed CLEM. We found that fluorescently detected mCherry-IL-2/LAMP1-EGFP vesicle pairs could be resolved as two tethered vesicles with a physical attachment in the higher-magnification EM images (Fig. 2D, 2E). The distance between these tethered vesicles is 26.6 ± 3.0 nm (n = 10), which is within the range of MCSs to define interorganelle tethering (9, 10). In addition, small regions of electron-dense accumulations, which are characteristic of MCSs (33) and are thought to represent protein tethering complexes, can be discerned at the site of contact between lysosomes and CytVs (Fig. 2E, arrowheads). Thus, together, these data demonstrate that CytVs can physically tether with LAMP1⁺ organelles.

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FIGURE 2.

IL-2⁺ vesicles are tethered to LAMP1⁺ vesicles. (A–C) IL-2⁺ vesicles are juxtaposed to LAMP1⁺ vesicles. Endogenous IL-2 and LAMP1 were stained with Alexa Fluor 647–labeled anti-human IL-2 mAb and Alexa Fluor 488–labeled anti-human LAMP1 mAb. The tethering rate of IL-2⁺ vesicles with LAMP1⁺ organelles and the total number of IL-2⁺ vesicles are shown in (B) and (C), respectively. The results for 90, 120, and 150 min were from five donors (79 cells), five donors (75 cells), or four donors (64 cells), respectively. (D and E) IL-2⁺ vesicles physically tether with LAMP1⁺ vesicles. CD4⁺ T cells were cotransfected with mCherry-IL-2 and LAMP1-EGFP. CLEM was performed to examine the localization of IL-2⁺ vesicles (red) and LAMP1⁺ lysosomes (green). Arrowheads highlight the small regions of electron-dense accumulations. Three examples of tethered IL-2–lysosome pairs are shown. Two exemplary cells from two independent experiments are shown. Scale bars, 5 μm (A and D) or 500 nm (E). **p < 0.01, ***p < 0.001.

Tethered CytV–lysosome pairs are transported in tandem and released sequentially

To exclude the possibility that tethering is an artifact introduced by fixation, we examined tethering in live CD4⁺ T cells by live-cell imaging. To be able to track the tethered TNF-α–LAMP1 pairs for a considerably long period of time, we used TIRF microscopy. With TIRF, only a thin layer of around 200 nm above the glass coverslip is illuminated. We observed that tethered TNF-α–LAMP1 pairs were transported in tandem (Fig. 3A, Supplemental Video 1). Remarkably, on rare occasions, we observed that after a short contact with another TNF-α–lysosome pair, the transport of a tethered TNF-α–lysosome pair could be switched to the opposite direction (Fig. 3B, Supplemental Video 2). Next, we examined the cotransport of IL-2–LAMP1 pairs using laser confocal scanning microscopy (LCSM). Live-cell imaging shows that tethered IL-2 and LAMP1 vesicles were also transported in a tandem manner (Fig. 3C, Supplemental Video 3). Very occasionally, separation of CytV–LAMP1 pairs was detected (Fig. 3D, Supplemental Video 4). Thus, we demonstrate that CytV–lysosome tethering is present in live cells and is not an artifact of fixation.

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FIGURE 3.

Cotransportation and sequential release of tethered CytV–LAMP1⁺ vesicles. LAMP1-EGFP and mCherry-TNF-α or mCherry-IL-2 as indicated were cotransfected in primary human CD4⁺ T cells. (A and B) Tethered TNF-α–LAMP1⁺ vesicles were transported in tandem. (C) Cotransportation of tethered IL-2–LAMP1⁺ vesicles. (D) Separation of tethered IL-2–LAMP1⁺ vesicles. (E and F) Sequential secretion of tethered TNF-α–LAMP1⁺ vesicles. The frame, at which TNF-α⁺ or LAMP1⁺ vesicles was released, is marked by red or green lines. Increase in LAMP1 fluorescence at the TIRF plane after TNF-α fusion is highlighted by the green boxes. (G and H) Two examples of simultaneous release of colocalized TNF-α and LAMP1. Transportation and release of vesicles were visualized at room temperature using either TIRF with a ×100 objective with an interval of 146 ms (A, B, and E–H) or with laser scanning confocal microscopy with an interval of 1 s (C and D). The CD4⁺ T cells were settled either on anti-CD3/anti-CD28 Ab-coated coverslips (A, B, and E–H) or on poly-l-ornithin–coated coverslips (C and D). All results are from three independent experiments. Scale bar, 1 μm.

We next examined if and how CytV–lysosome pairs are released. We examined TNF-α–lysosome pairs with high frequency time-lapse imaging (∼7 Hz) using TIRF. We found that after docking at the plasma membrane, TNF-α fluorescence vanished first followed by the fusion of the tethered lysosome (Fig. 3E, 3F, Supplemental Videos 5 and 6). Notably, after the release of TNF-α was initiated, as indicated by a sharp drop of the mCherry (TNF-α) fluorescence, the fluorescence intensity of LAMP1⁺ vesicles at the TIRF plane was first intensified (highlighted by the light green boxes) and then gradually declined with time (Fig. 3E, 3F). The increase in fluorescence of the second vesicle suggests that the vesicle moves closer to the TIRF plane because the intensity of illumination by the evanescent field would increase exponentially with the distance (34). Thus, these results indicate that tethered TNF-α⁺ and LAMP1⁺ vesicles are released sequentially. Notably, as shown in these two examples, TNF-α and LAMP1 fluorescence often appeared to be highly colocalized, which could be the result of a quasiperpendicular spatial orientation of the tethered TNF-α–LAMP1 pairs with respect to the plasma membrane. More precisely speaking, as sketched in Fig. 3E, 3F, if LAMP1⁺ vesicles are positioned on top of the TNF-α⁺ vesicles, when the latter is docked at the plasma membrane, the fluorescence from TNF-α⁺ and LAMP1⁺ vesicles would be largely overlapped when observed at the TIRF plane.

To test this hypothesis, we needed to be able to distinguish simultaneous release from sequential release. The former would be the consequence of the release of truly colocalized TNF-α⁺ and LAMP1⁺ vesicles. In transfected CD4⁺ T cells, colocalization of TNF-α–LAMP1 was indeed observed on some occasions. Especially when the overexpression level of LAMP1 was high, TNF-α was observed to be surrounded by LAMP1. This colocalization of overexpressing TNF-α and LAMP1 can be resulted from missorted overexpressing LAMP1; as such, TNF-α–LAMP1 colocalization was never observed in endogenous conditions. Nevertheless, we took advantage of those colocalized TNF-α–LAMP1 vesicles to characterize simultaneous release of TNF-α and LAMP1. We found that in the case of simultaneous release, the intensity of LAMP1 fluorescence did not increase after TNF-α release (Fig. 3G, 3H). Therefore, we prove that the sequential releases shown in Fig. 3E, 3F hold true, which further supports our conclusion that TNF-α⁺ vesicles and LAMP⁺ endocytic organelles are tethered.

Tethering between CytVs and endocytic organelles is mediated by Vti1b

To determine the molecular basis mediating the tethering between CytVs and lysosomes, we put our focus on Vti1b, a SNARE protein necessary for the juxtaposition of LG and CD3⁺ endosomes in CD8⁺ T cells (16). We first used modified siRNA to downregulate endogenous Vti1b in primary human CD4⁺ T cells. We show that expression of Vti1b in CD4⁺ T cells was downregulated by modified siRNA to <40% at both mRNA and protein levels (Fig. 4A–C). Immunostaining shows that in Vti1b-downregulated CD4⁺ cells, the fraction of TNF-α⁺ vesicles tethered with lysosomes (LAMP1⁺) was significantly reduced (Fig. 4D, 4E). The total numbers of TNF-α⁺ or LAMP1⁺ vesicles remained unchanged (Fig. 4F). These experiments show that Vti1b is involved in tethering TNF-α⁺ vesicles to lysosomes and that TNF-α–lysosome tethering does not affect the number of TNF-α⁺ vesicles and lysosomes. We further examined whether blockage of Vti1b function could affect TNF-α release. Because Vti1b siRNA is not fluorescently labeled, to identify transfected T cells, we overexpressed the cytosolic fragment of Vti1b 1–207 to block the interaction of Vti1b with its cognate partners (35). We examined the secretion of TNF-α by TIRF microscopy. The results show that the cytosolic fragment of Vti1b 1–207 significantly diminished the fraction of the T cells that could release TNF-α (Fig. 4G) as well as the fusion events in each TNF-α–secreting cell (Fig. 4H). This result indicates that Vti1b-regulated LAMP1 tethering is also involved in TNF-α secretion.

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FIGURE 4.

CytV–endocytic organelle tethering is Vti1b dependent. (A–C) Downregulation of Vti1b in T cells. Primary human CD4⁺ T cells were stimulated with anti-CD3/anti-CD28 Ab-coated beads. Then, modified siRNA against Vti1b or control (Ctrl) siRNA were introduced into the cells. The cells were used for analysis 36 h after transfection. The mRNA level of Vti1b was determined using quantitative RT-PCR (n = 3 independent experiments with duplicates for each condition) in (A). TBP was applied as the reference gene. The protein level of Vti1b was determined using Western blotting with γ-tubulin as the internal control as shown in (B). Quantification of (B) is shown in (C) (n = 3 independent experiments). (D) Tethering between TNF-α⁺ and LAMP1⁺ vesicles is decreased by Vti1b downregulation. Stimulated primary human CD4⁺ T cells were conjugated with target cells (SEA/SEB-pulsed Raji cells) at 37°C for 60 min and then fixed. Quantification of tethered TNF-α fraction as well as the total vesicle numbers of TNF-α⁺ and LAMP1⁺ vesicles (30 cells from three independent experiments) are shown in (E) and (F). (G and H) Overexpression of Vti1b 1–207 reduces TNF-α release. Stimulated primary human CD4⁺ T cells transiently overexpressed mCherry-TNF-α with Vti1b FL-TFP or Vti1b 1-207-TFP were settled on anti-CD3/anti-CD28 Ab-coated coverslips. Movement and release of vesicles at the IS at 37°C were visualized by TIRF microscopy. The percentage of CD4⁺ T cells that could release TNF-α is shown in (G). The number of TNF-α released in each cell is shown in (H). Results are from five independent experiments. (I–K) Tethering between IL-2 and LAMP1 is decreased by Vti1b downregulation. Stimulated primary human CD4⁺ T cells were settled on anti-CD3/anti-CD28 Ab-coated coverslips at 37°C for 150 min before fixation. Endogenous IL-2 and LAMP1 were stained. The IS planes are shown, whereby IL-2/LAMP1 pairs are highlighted by the arrowheads. Percentage of tethered IL-2⁺ vesicles and the vesicle numbers from three independent experiments are shown in (J) and (K). All samples were scanned with deconvolution microscopy with a ×63 objective. Scale bar, 5 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

Similarly, the level of IL-2–lysosome tethering is also decreased significantly by downregulation of Vti1b (Fig. 4I, 4J). The number of IL-2⁺ vesicles remained unchanged, whereas the number of LAMP1⁺ vesicles was slightly reduced (Fig. 4K), which cannot be fully attributed to the substantial reduction in the level of IL-2–lysosome tethering by Vti1b downregulation. Thus, we conclude that tethering between CytVs and lysosomes is mediated by Vti1b.

Next, we looked for the interaction partner of Vti1b to regulate tethering. We focused on EpsinR because it has been shown to bind Vti1b and regulate the sorting of Vti1b (23). Using co-IP, we confirmed that two point mutants of human Vti1b, namely Vti1b-F73S and Vti1b-E12A, were not able to bind endogenous EpsinR as their WT counterpart Vti1b-WT (Fig. 5A), which is in good agreement with the previous report (23). Overexpression of Vti1b-F73S or Vti1b-E12A in primary CD4⁺ T cells substantially reduced TNF-α–lysosome tethering (Fig. 5B, 5C) without affecting the total number of TNF-α⁺ vesicles or lysosomes (Fig. 5D). We noticed that the overexpressed Vti1b is predominantly distributed on the plasma membrane, which might be due to the redundancy introduced by overexpression.

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FIGURE 5.

Interaction between Vti1b and EpsinR is required for CytV–endocytic organelle tethering. (A) Point mutants E12A and F73S of Vti1b abolish the interaction with EpsinR. GFP-tagged Vti1b WT, F73S, E12A, and GFP control vector were transiently transfected in HEK293T cells. Cells were lysed 24 h after transfection for co-IP. (B) Point mutants E12A and F73S of Vti1b substantially reduce tethering between TNF-α and LAMP1. Fluorescent protein mCherry-tagged Vti1b WT, F73S, or E12A were transiently overexpressed in primary human CD4⁺ T cells. Twenty-four hours after transfection, T cells were settled on anti-CD3/anti-CD28 Ab-coated coverslips for 60 min followed by staining of endogenous TNF-α and LAMP1. The samples were scanned with deconvolution microscopy with a ×63 objective. A few examples of TNF-α–LAMP1 pairs are highlighted by arrowheads. Scale bar, 5 μm. Quantification of tethering probability and numbers of TNF-α⁺ and LAMP1⁺ vesicles are shown in (C) and (D), respectively. All results are from three independent experiments. ***p < 0.001

Our next question is whether these two point mutants of Vti1b (F73S and E12A) may interfere with the interaction between Vti1b and its cognate SNARE proteins. To this end, because it is very difficult to get a large enough number of primary T cells required to perform co-IP for all SNARE partners of Vti1b, we sought assistance from molecular modeling. We tested three computational tools, MAESTRO, ROSETTA, and BeAtMuSiC (36), based on structural data on the Vti1b/EpsinR complex. BeAtMuSiC (31) was determined as a suitable tool as it predicts seven out of eight residues that were previously experimentally determined to be important for the formation of this complex (Supplemental Table I). Furthermore, BeAtMuSiC suggests that F73 and E12 are not involved in Vti1b binding with its cognate SNARE proteins Syntaxin7, Syntaxin8, and VAMP8/Endobrevin (Supplemental Table II). Therefore, Vti1b F73S and Vti1b E12A appear to specifically block interaction between Vti1b and EpsinR without affecting the binding of Vti1b to its cognate SNARE partners. We therefore conclude that the interaction between Vti1b and EpsinR is required for CytV–lysosome tethering.

Lysosome tethering determines the direction of CytV transport

One important question is whether lysosome tethering contributes in any way to the distinct destination of TNF-α⁺ and IL-2⁺ vesicles, and if yes, how. To address this question, we designed experiments to analyze the localization of tethered versus untethered TNF-α⁺ and IL-2⁺ vesicles. For this analysis, the whole cell was divided into two different segments along the z-direction. As shown in Fig. 6A, the segment encompassing one fourth of the cell’s diameter close to the coverslip was defined as the IS area, and the rest of the cell was defined as the distal area. The analysis shows that the majority of lysosome-tethered TNF-α ⁺ vesicles were in the distal area, whereas the majority of untethered TNF-α⁺ vesicles were close to the IS (Fig. 6B, 6C). Conversely, the majority of lysosome-tethered IL-2 were enriched at the IS, whereas the majority of untethered IL-2 were in the distal area (Fig. 6B, 6D). These data suggest that by tethering to lysosomes, CytVs can be delivered to their desired destinations for both polarized and multidirectional localization.

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FIGURE 6.

Tethering to LAMP1⁺ organelles determines the transport direction of cytokines. CD4⁺ T cells were restimulated with anti-CD3/anti-CD28 Ab-coated beads for 120 min (B–D) or 90 or 120 min (E and F). After bead removal, the T cells were fixed and stained with corresponding Abs. (A) Distribution of vesicles in the vicinity to the IS (@IS) or away from the IS (@distal). (B–D) Tethering is involved in directional transport for both TNF-α⁺ and IL-2⁺ vesicles. Anti–TNF-α or anti–IL-2 and anti-LAMP1 Abs were used. Z-stacks were taken with a z-step size of 0.2 μm by deconvolution microscopy with a ×63 objective. Distribution for TNF-α⁺ and IL-2⁺ vesicles at the IS or distal areas was shown in (B). Analyses of distribution of TNF-α⁺ or IL-2⁺ vesicles are shown in (C) and (D). Scale bar, 5 μm. Results were from three independent experiments (24 cells). (E and F) Tethering of LAMP1 with CytVs in the same T cells. Endogenous TNF-α, IL-2, and LAMP1 were stained after fixation. The fractions of LAMP1 tethered with IL-2, TNF-α, or IL-2 and TNF-α are shown in (E). The numbers of CytVs tethered with LAMP1⁺ organelles are shown in (F). Results were from two independent experiments (16 cells). **p < 0.01, ***p < 0.001. MIP, maximum intensity projection.

The next question is whether the same LAMP1⁺ organelle can tether with both TNF-α ⁺ and IL-2⁺ vesicles. To address this question, we stained endogenous TNF-α, IL-2, and LAMP1 simultaneously in the same T cells. Quantifications shows that for both time points (90 and 120 min), the fractions of LAMP1⁺ vesicles tethered with IL-2⁺ or TNF-α⁺ vesicles were comparable (Fig. 6E, IL-2 versus TNF-α). We also observed that only occasionally, the same LAMP1⁺ vesicles could tether with both IL-2⁺ and TNF-α⁺ vesicles (Fig. 6F, IL-2 and TNF-α). Moreover, the numbers of IL-2⁺ or TNF-α⁺ vesicles that tethered with LAMP1 also confirm that the majority of TNF-α⁺ and IL-2⁺ vesicles do not share the same LAMP1⁺ organelles (Fig. 6F).

Proportion of endocytic organelle-tethered CytVs modulates transport mode of CytVs

Subsequently, we investigated how lysosome tethering regulates the two modes of distribution. The direction of cargo transport is primarily defined by the attached motor proteins, namely plus and minus end directed microtubule motor proteins kinesin and dynein. There are more than 40 members of the kinesin superfamily (37). Specifically, blocking the function of several kinesin isoforms simultaneously either by pharmaceutical intervention or by small interference RNA in primary human T cells is experimentally very challenging, if not impossible. Therefore, we used mathematical models to understand how tethering could regulate these two modes of cytokine transport. First, we quantified the experimental observations by the introduction of various probabilities for untethered (index 0) and tethered (index t) vesicles. The probabilities for dynein and kinesins being attached to tethered and untethered TNF-α or IL-2 vesicles and the probabilities for untethered and tethered TNF-α⁺ or IL-2⁺ vesicles to be located at the IS or distal to it were extracted from the experimental data (Figs. 6, 7) and are listed in Supplemental Table III. Based on these results, we analyzed the interrelation between lysosome tethering and location probabilities (close to or away from the IS) for TNF-α⁺ and IL-2⁺ vesicles, respectively.

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FIGURE 7.

Association of dynein and kinesin on TNF-α⁺ and IL-2⁺ vesicles. Anti–TNF-α (A) or anti–IL-2 (C, green), anti-LAMP1 (red), and anti-kinesin or anti-dynein (magenta) Abs were used. Images were acquired by SIM using a ×63 objective. The association between TNF-α and motor proteins was quantified in (B). Ten tethered or untethered TNF-α⁺ vesicles were randomly selected from each cell [23 cells from three independent experiments (B)]. Ten tethered or untethered IL-2⁺ vesicles were randomly selected from each cell (25 cells from three independent experiments), and the association between IL-2 and motor proteins is shown in (D). Scale bar, 5 μm. ***p < 0.001

As shown in Eq. 1, the probability for CytV to be located at the IS () is the sum of the probabilities of tethered and untethered CytV that are located at the IS:(1)The probabilities of tethered and untethered CytV that are located at the IS ( and ) were experimentally determined. For TNF-α, =0.3 (=0.7), and 0.6 (=0.4). For IL-2, =0.4 (=0.6), and = 0.8 (=0.2). Therefore, for TNF-α and IL-2, the correlation between and is opposite, as elaborated in Eqs. 2 and 3:(2)(3)The fraction of TNF-α accumulated at the IS is negatively correlated with the probability of TNF-α tethering with lysosomes () (Fig. 8A), whereas for IL-2, the higher the tethering rate, the higher the propensity to stay at the IS (Fig. 8B). For the vesicle distribution to be considered to be multidirectional, at the most, half of the vesicles may be found near the IS. Thus, a line may be drawn when equals 0.5, which defines the boundary between polarized versus multidirectional transport. Consequently, the tethering probability of TNF-α must be larger than 0.33 (Fig. 8A, gray area) for multidirectional transport to occur. For IL-2, to achieve a polarized pattern (), the tethering probability of IL-2 needs only to be larger than 25% (Fig. 8B, gray area). Thus, we conclude that tethering with lysosomes facilitates that TNF-α and IL-2 achieve their desired destinations.

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FIGURE 8.

Mathematical models for endocytic organelle tethering directed cytokine transport. (A and B) The fractions of CytVs accumulated at the IS and the proportion of lysosome-tethered CytVs are negatively correlated for TNF-α⁺ vesicles and positively correlated for IL-2⁺ vesicles. The gray area marks the fraction of tethered vesicles required for TNF-α to have a multidirectional distribution (A) or for IL-2 to have an IS-polarized distribution (B). (C) The correlation of the probability to find IL-2 vesicles at the IS (P_IS) with the attached kinesins (P_kin) when dynein outperforms kinesin (Mechanism 1). The dashed lines show the required P_kin to meet experimentally determined P_IS. (D) Distribution of IL-2 when kinesin is inhibited (Mechanism 2). The probability of kinesin inhibition () was set to 0, 0.5, 0.75, and 1 for Eqs. 6 and 7 (left panel). To obtain the experimentally determined (0.4) and (0.8), the correlation between and P_kin is shown in the right panel. (E) Distribution of IL-2 with the assumptions that kinesin is partially inhibited and that dynein outperforms uninhibited kinesin (Mechanism 3). (F) The correlation between and P_kin to obtain the experimentally determined (0.4) and (0.8) with Mechanism 3.

Next, we analyzed the impact of tethering-regulated motor protein recruitment on CytV transport. For TNF-α, we assume that the kinesin outperforms dynein if both motor proteins are recruited on the same vesicle, which is also supported by the fact that the stall force of kinesin is much higher than the stall force of dynein (38). We also introduced a dynein inhibition probability on TNF-α vesicles and denoted it as . Then, the probability for untethered TNF-α to be at the IS, , is:where the first term on the right-hand side is the probability for having neither dynein nor kinesin on untethered TNF-α vesicles, in which case the vesicle stays close to the microtubule organizing center (MTOC) and thus at the IS. The second term is the probability that a vesicle contains dynein, that this dynein is not inhibited, and that the vesicle does not contain kinesin, in which case it would also stay near the MTOC/IS. The third term is the probability that dynein is present but is inhibited, yet kinesin is also absent, in which case the vesicle would still remain near the IS. This equation can be simplified to the following:Notably, neither nor play a role in determination of TNF-α distribution, only the presence of kinesin. In other words, for an untethered TNF-α⁺ vesicle to remain at the IS, kinesin has to be absent, and for their transport away from the IS, kinesin must be present. Because the quantification of our experimental observation about the number of vesicles being located at the IS or at the distal area (dist) shows that is around 0.6 (and hence = 0.4), there should be other kinesins (e.g., kinesin-3 [denoted as kin-x]) involved besides kinesin-1, so that we haveBecause we measured the probability of finding kinesin on untethered vesicles to be and , the probability for other kinesins, , should be around 0.2. In comparison, on tethered TNF-α⁺ vesicles, the probability to have dynein or kinesin is = 0.4 and = 0.5, respectively. The resulting probability to find tethered TNF-α⁺ vesicles at the IS are then:(4)Experimentally, we observed , which is smaller than the value predicted from Eq. 4 based on the measurement of kinesin-1. Therefore, we postulate again the presence of other kinesins on tethered TNF-α⁺ vesicles, such thatTherefore, we get the following:To obtain , one must again have , which is similar to , the probability for other kinesins on untethered TNF-α⁺ vesicles.

To analyze how the recruitment of motor proteins and tethering contributes to the final distribution of IL-2, we first assumed that kinesin outperforms dynein, as for TNF-α. Thus:With this, the probability for kinesin on untethered and tethered IL-2 should be = 0.6 and = 0.2. The latter is incompatible with our experimental result = 0.4. Therefore, the assumption that kinesin always outperforms dynein must be false for IL-2. We can postulate two alternative scenarios that could help explain this discrepancy.

First, we could assume conversely that dynein outcompetes kinesin (Mechanism 1) (e.g., by the number of dyneins being much larger than the number of kinesins). Then, we obtainwhere the first term on the right-hand side is the probability for the absence of kinesin, and the second term is the probability that both dynein and kinesin are present, in which case dynein wins. By inserting the experimental values for (0.3) and (0.5), we get:(5)(6)Fig. 8C shows the dependency of IS enrichment (P_IS) on the attachment of kinesin (P_kin), as given by Eqs. 5 and 6. One can see that the probability to find IL-2 at the IS is negatively correlated with their attached kinesin (Fig. 8C), which is expected. In addition, one can see that by increasing the deposition of dynein, tethering of IL-2⁺ vesicles increases the propensity of finding IL-2⁺ vesicles at the IS. For the probability of untethered IL-2 being found at the IS, , to be around 0.4 (experimental observation), the required should be 0.86 (Fig. 8C, blue dashed lines), which again implies additional kinesins are involved in the case of untethered IL-2⁺ vesicles. For tethered IL-2⁺ vesicles, = 0.8, and the corresponding should be 0.4 (Fig. 8C, red dashed lines), which agrees with the experimentally determined (Fig. 7, Supplemental Table III).

In a second scenario, we assumed that kinesin is inhibited on IL-2 (Mechanism 2) with probability . Then, one has(7)(8)where the first term on the right-hand side is the probability that kinesin is absent on IL-2⁺ vesicles, in which case the vesicles stay close to the MTOC and thus at the IS. The second term is the probability that kinesins are present but inhibited. By plotting P_IS as a function of , we find that with Mechanism 2, the probability of IL-2⁺ vesicles to be enriched at the IS is positively correlated with and proportional to (Fig. 8D, left panel) and independent of dynein attachment (Eqs. 7, 8). To reproduce the experimentally determined values for (0.4) and (0.8), the kinesin inhibition probability on tethered IL-2 must be substantially higher compared with untethered if P_kin is identical for tethered and untethered IL-2. If, in contrast, is identical in both cases, the recruitment of kinesin on tethered IL-2 should be considerably lower than untethered IL-2 (Fig. 8D, right panel).

The above-mentioned Mechanisms 1 and 2 could also be combined (kinesin inhibition and outperformance of dynein over uninhibited kinesin, Mechanism 3). Then, one has the following:In this combined case, the probability of IS enrichment of IL-2⁺ vesicles is also positively correlated with and proportional to and, in addition, is also dependent on dynein attachment (Fig. 8E). To obtain the experimentally determined values for (0.4) and (0.8), the dependency of on P_kin is similar as for Mechanism 2, namely identical P_kin requires higher on tethered compared with untethered IL-2⁺ vesicles, and identical would imply a lower level of total kinesin on tethered as compared with untethered IL-2⁺ vesicles (Fig. 8F). Notably, the range of P_kin to obtain a positive value of from Mechanism 3 is narrower than Mechanism 2. In addition, Mechanism 2 and Mechanism 3 predict higher and/or lower P_kin on tethered IL-2⁺ vesicles, suggesting that tethering is correlated with removing or detaching kinesins on tethered IL-2⁺ vesicles.