From 01d02de765797c9d0b5c74bb8f7ba4cd631ad70a Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Wed, 8 Sep 2021 23:43:16 -0400 Subject: [PATCH] ENH proof aim 2b --- tables/integrin_1_reg.tex | 4 +- tables/integrin_2_reg.tex | 2 +- tex/thesis.tex | 230 ++++++++++++++++++-------------------- 3 files changed, 112 insertions(+), 124 deletions(-) diff --git a/tables/integrin_1_reg.tex b/tables/integrin_1_reg.tex index 1e963cf..ffc769a 100644 --- a/tables/integrin_1_reg.tex +++ b/tables/integrin_1_reg.tex @@ -3,13 +3,13 @@ \begin{tabular}{@{\extracolsep{5pt}}lcc} \\[-1.8ex]\hline \hline \\[-1.8ex] -\\[-1.8ex] & \ptmemp{} \% & \pthp{} \\ +\\[-1.8ex] & \ptmemp{} & \pthp{} \\ \hline \\[-1.8ex] CD49a & 0.034 & 34.500 \\ CD49b & 0.024 & 397.000 \\ Constant & 0.313$^{***}$ & 1,233.250$^{***}$ \\ \hline \\[-1.8ex] -Observations & 8 & 8 \\ +% Observations & 8 & 8 \\ R$^{2}$ & 0.222 & 0.270 \\ Adjusted R$^{2}$ & $-$0.089 & $-$0.022 \\ \hline diff --git a/tables/integrin_2_reg.tex b/tables/integrin_2_reg.tex index 6763c7e..a7593aa 100644 --- a/tables/integrin_2_reg.tex +++ b/tables/integrin_2_reg.tex @@ -9,7 +9,7 @@ CD49b & 0.007 & $-$0.015 \\ Constant & 0.105$^{***}$ & 0.272$^{***}$ \\ \hline \\[-1.8ex] -Observations & 12 & 12 \\ +% Observations & 12 & 12 \\ R$^{2}$ & 0.082 & $-$0.153 \\ \hline \hline \\[-1.8ex] diff --git a/tex/thesis.tex b/tex/thesis.tex index d957571..e760269 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -3668,8 +3668,6 @@ interest using \glspl{mab}. \subsection{DMSs Temporal Modulation} -% METHOD The concentration for the surface marker cleavage experiment was much -% higher, if that matters \glspl{dms} were digested in active T cell cultures via addition of sterile \product{\gls{colb}}{\sigald}{11088807001} or \product{\gls{cold}}{\sigald}{11088858001}. Collagenase was dissolved in @@ -3680,9 +3678,9 @@ media normally used to feed the cells during the regular media addition cycle at day 4. Cultures were then incubated as described in \cref{sec:tcellculture}, and the \glspl{dms} were verified to have been digested after \SI{24}{\hour}. -Adding \glspl{dms} was relatively much simpler; the number of \gls{dms} used per -area on day 0 was scaled up by 3 on day 4 to match the change from a 96 well -plate to a 24 well plate, effectively producing a constant activation signal. +Adding \glspl{dms} was simpler; the number of \gls{dms} used per area on day 0 +was scaled up by 3 on day 4 to match the change from a 96 well plate to a 24 +well plate, effectively producing a constant activation signal. \subsection{Mass Cytometry and Clustering Analysis} @@ -3692,10 +3690,10 @@ used according to the manufacturer’s instructions. \numrange{2e6}{3e6} stained cells per group were analyzed on a Fluidigm Helios. Unbiased cell clusters were obtained using \gls{spade} analysis by pooling three -representative \gls{fcs} files and running the \gls{spade} pipeline with k-means -clustering (k = 100), arcsinh transformation with cofactor 5, density -calculation neighborhood size of 5 and local density approximation factor of -1.5, target density of 20000 cells, and outlier density cutoff of +representative \gls{fcs} files and running \gls{spade} with k-means clustering +(k = 100), arcsinh transformation with cofactor 5, density calculation +neighborhood size of 5, local density approximation factor of 1.5, target +density of 20000 cells, and outlier density cutoff of \SI{1}{\percent}\cite{Qiu2017}. All markers in the \gls{cytof} panel were used in the analysis @@ -3716,7 +3714,7 @@ analyzing via a \bd{} Accuri flow cytometer. To block the \gls{il15r}, we supplemented T cell cultures activated with \gls{dms} with either -\product{\anti{\gls{il15r}}}{Rnd}{AF247} or \product{\gls{igg} isotype +\product{\anti{\gls{il15r}}}{RnD}{AF247} or \product{\gls{igg} isotype control}{RnD}{AB-108-C} at the indicated timepoints and concentrations. T cells were grown as otherwise described in \cref{sec:tcellculture} with the exception that volumes were split by $\frac{1}{3}$ to keep the culture volume @@ -3739,11 +3737,11 @@ porcine-derived collagen, this enzyme should target the \gls{dms} while sparing the cells along with any markers we wish to analyze. We tested this specific hypothesis using either \gls{colb}, \gls{cold} or \gls{hbss}, and stained the cells using a typical marker panel to assess if any of the markers were cleaved -off by the enzyme which would bias our final readout. We observed that the -marker histograms in the \gls{cold} group were similar to that of the buffer -group, while the \gls{colb} group visibly lowered CD62L and CD4, indicating -partial enzymatic cleavage (\cref{fig:collagenase_fx}). Based on this result, we -used \gls{cold} moving forward. +off by the enzyme which would bias our final readout. The marker histograms in +the \gls{cold} group were similar to that of the buffer group, while the +\gls{colb} group visibly lowered CD62L and CD4, indicating partial enzymatic +cleavage (\cref{fig:collagenase_fx}). Based on this result, we used \gls{cold} +moving forward. \begin{figure*}[ht!] \begingroup @@ -3763,13 +3761,13 @@ inhibited in the \textit{added} group while the cells seemed to grow faster in the \textit{removed} group relative to the \textit{no change} group (\cref{fig:add_rem_growth}). Additionally, the \textit{removed} group seemed to have a negative growth rate in the final \SI{4}{\day} of culture, indicating -that either the lack activation signal had slowed the cell growth down or that -the cells were growing fast enough to outpace the media feeding schedule. The -viability was the same between all groups, indicating that this negative growth -rate and the lower growth rate in the \textit{added} group were likely not due -to cell death (\cref{fig:add_rem_viability}). Interestingly, the \textit{added} -group had significantly higher \pth{} cells compared to the \textit{no change} -group, and the inverse was true for the \textit{removed} group +that either the lack activation signal had slowed cell growth or that the cells +were growing fast enough to outpace the media feeding schedule. The viability +was the same between all groups, indicating that this negative growth rate and +the lower growth rate in the \textit{added} group were likely not due to cell +death (\cref{fig:add_rem_viability}). Interestingly, the \textit{added} group +had significantly higher \pth{} cells compared to the \textit{no change} group, +and the inverse was true for the \textit{removed} group (\cref{fig:add_rem_cd4}). These results show that the growth rate and phenotype are fundamentally altered by changing the number of \glspl{dms} temporally. @@ -3838,33 +3836,33 @@ We next asked what the effect of removing the \glspl{dms} would have on other phenotypes, specifically \gls{tcm} and \gls{tscm} cells. To this end we stained cells using a 34-marker mass cytometry panel and analyzed them using a Fluidigm Helios. After pooling the \gls{fcs} file events from each group and analyzing -them via \gls{spade} we see that there is a strong bifurcation of CD4 and CD8 T -cells. We also observe that among CD27, CD45RA, and CD45RO (markers commonly -used to identify \gls{tcm} and \gls{tscm} subtypes) we see clear `metaclusters' -composed of individual \gls{spade} clusters which are high for that marker +them via \gls{spade} we saw a strong bifurcation of CD4 and CD8 T cells. When +looking at CD27, CD45RA, and CD45RO (markers commonly used to identify \gls{tcm} +and \gls{tscm} subtypes) we saw clear ``metaclusters'' composed of individual +\gls{spade} clusters which are high for these markers (\cref{fig:spade_msts,fig:spade_gates}). We then gated each of these metaclusters according to their marker levels and assigned them to one of three phenotypes for both the CD4 and CD8 compartments: \gls{tcm} (high CD45RO, low CD45RA, high CD27), \gls{tscm} (low CD45RO, high CD45RA, high CD27), and -`transitory' \gls{tscm} cells (mid CD45RO, mid CD45RA, high CD27). Together +``transitory'' \gls{tscm} cells (mid CD45RO, mid CD45RA, high CD27). Together these represent low differentiated cells which should be highly potent as anti-tumor therapies. When quantifying the number of cells from each experimental group in these -phenotypes, we clearly see that the number of lower differentiated cells is much -higher in the \textit{no change} or \textit{removed} groups compared to the -\textit{added} group (\cref{fig:spade_quant}). Furthermore, the \textit{removed} -group had a much higher fraction of \gls{tscm} cells compared to the \textit{no - change} group, which had more `transitory \gls{tscm} cells'. The majority of -these cells were \cdp{8} cells. When analyzing the same data using \gls{tsne}, -we observe a higher fraction of CD27 and lower fraction of CD45RO in the the +phenotypes, the number of lower differentiated cells was much higher in the +\textit{no change} or \textit{removed} groups compared to the \textit{added} +group (\cref{fig:spade_quant}). Furthermore, the \textit{removed} group had a +much higher fraction of \gls{tscm} cells compared to the \textit{no change} +group, which had more ``transitory \gls{tscm} cells.'' The majority of these +cells were \cdp{8} cells. When analyzing the same data using \gls{tsne}, we +observed a higher fraction of CD27 and lower fraction of CD45RO in the \textit{removed} group (\cref{fig:spade_tsne_all}). When manually gating on the CD27+CD45RO- population, we see there is higher density in the \textit{removed} group, indicating more of this population (\cref{fig:spade_tsne_stem}). Together, these data indicate that removing \glspl{dms} at lower timepoints -leads to potentially higher expansion, lower \pthp{}, and higher fraction of -lower differentiated T cells such as \gls{tscm}, and adding \gls{dms} seems to -do the inverse. +leads to higher expansion, lower \pthp{}, and higher fraction of +lower differentiated T cells such as \gls{tscm}, and adding \gls{dms} does the +inverse. \subsection{Blocking Integrin Does Not Alter Expansion or Phenotype} @@ -3872,8 +3870,8 @@ One of the reasons the \gls{dms} platform might perform better than the beads is the fact that they are composed of gelatin, which is a collagen derivative. The beads are simply \gls{mab} attached to a polymer resin coated onto an iron oxide core, and thus have no analogue for collagen. Collagen domains present on the -\gls{dms} group could be creating pro-survival and pro-expansion signals to the -T cells through \gls{a2b1} and \gls{a2b2}, causing them to grow better in the +\gls{dms} group could provide pro-survival and pro-expansion signals to the T +cells through \gls{a2b1} and \gls{a2b2}, causing them to grow better in the \gls{dms} system. \begin{figure*}[ht!] @@ -3907,14 +3905,14 @@ T cells through \gls{a2b1} and \gls{a2b2}, causing them to grow better in the We tested this hypothesis by adding blocking \glspl{mab} against \gls{a2b1} and/or \gls{a2b2} to running T cell cultures activated using the \glspl{dms}. -These block \glspl{mab} were added at day 6 of culture when \gls{a2b1} and -\gls{a2b2} were known to be expressed\cite{Hemler1990}. We found that the fold -expansion was identical in all the blocked groups vs the unblocked control group -(\cref{fig:inegrin_1_fc}). Furthermore, we observed that the \ptmemp{} (total -and across the CD4/CD8 compartments) was not significantly different between any -of the groups (\cref{fig:inegrin_1_mem,tab:integrin_1_reg}). We also noted that -\gls{a2b1} and \gls{a2b2} were present on the surface of a significant subset of -T cells at day 6, showing that the target we wished to block was present +These blocking \glspl{mab} were added at day 6 of culture when \gls{a2b1} and +\gls{a2b2} were known to be expressed\cite{Hemler1990}. The fold expansion was +identical between the blocked and unblocked groupds (\cref{fig:inegrin_1_fc}). +Furthermore, the \ptmemp{} (total and across the CD4/CD8 compartments) was not +significantly different between any of the groups +(\cref{fig:inegrin_1_mem,tab:integrin_1_reg}). Furthermore, \gls{a2b1} and +\gls{a2b2} were present on the surface of a significant subset of T cells at day +6, showing that the target we wished to block was present (\cref{fig:inegrin_1_cd49}). \begin{figure*}[ht!] @@ -3943,14 +3941,14 @@ T cells at day 6, showing that the target we wished to block was present \input{../tables/integrin_2_reg.tex} \end{table} -Since this last experiment gave a negative result, we decided to block +Since this initial experiment gave a negative result, we decided to block \gls{a2b1} and \gls{a2b2} harder by adding \glspl{mab} at more timepoints between day 0 and day 6, hypothesizing that the majority of the signaling would be during the period of culture where the \gls{dms} surface concentration was at -its maximum. Once again, we observed no difference between any of the blocked -conditions and the unblocked controls in regard to expansion -(\cref{fig:inegrin_2_fc}). Furthermore, none of the \ptmemp{} readouts (total, -CD4, or CD8) were statistically different between groups +its maximum. Once again, there was no difference between the blocked and +unblocked conditions in regard to expansion (\cref{fig:inegrin_2_fc}). +Furthermore, none of the \ptmemp{} readouts (total, CD4, or CD8) were +statistically different between groups (\cref{fig:inegrin_2_mem,tab:integrin_2_reg}). Taken together, these data suggest that the advantage of the \gls{dms} platform @@ -3959,11 +3957,11 @@ is not due to signaling through \gls{a2b1} or \gls{a2b2}. \subsection{Blocking IL15 Does Not Alter Expansion or Phenotype} \gls{il15} is a cytokine responsible for memory T cell survival and maintenance. -Furthermore, we observed in other experiments that it is secreted to a much -greater extend in \gls{dms} compared to bead cultures (\cref{fig:doe_luminex}). -One of our driving hypotheses in designing the \gls{dms} system was that the -higher cell density would lead to greater local signaling. Since we observed -higher \ptmemp{} across many conditions, we hypothesized that \gls{il15} may be +Furthermore, previous experiments showed that it is secreted to a much greater +extend in \gls{dms} compared to bead cultures (\cref{fig:doe_luminex}). One of +our driving hypotheses in designing the \gls{dms} system was that the higher +cell density would lead to greater local signaling. Since we observed higher +\ptmemp{} across many conditions, we hypothesized that \gls{il15} may be responsible for this, and further that the unique \textit{cis/trans} activity of \gls{il15} may be more active in the \gls{dms} system due to higher cell density. @@ -3980,7 +3978,7 @@ density. \endgroup \caption[IL15 Blocking I] {Blocking IL15Ra does not lead to differences in memory or growth. - \subcap{fig:il15_1_overview}{Experimental overview} + \subcap{fig:il15_1_overview}{Experimental overview}. Longitudinal measurements of \subcap{fig:il15_1_fc}{fold change} and \subcap{fig:il15_1_viability}{viability} for blocked and unblocked @@ -3993,7 +3991,7 @@ density. We first tested this hypothesis by blocking \gls{il15r} with either a specific \gls{mab} or an \gls{igg} isotype control at -\SI{5}{\ug\per\ml}\cite{MirandaCarus2005}. We observed no difference in the +\SI{5}{\ug\per\ml}\cite{MirandaCarus2005}. There was no difference in the expansion rate of blocked or unblocked cells (this experiment also had bead-based groups but they did not expand well and thus were not included) (\cref{fig:il15_1_fc}). Furthermore, there were no differences in viability @@ -4015,89 +4013,79 @@ the markers, and by extension showing no difference in phenotype \endgroup \caption[IL15 Blocking II] {Blocking soluble IL15 does not lead to differences in memory or growth. - \subcap{fig:il15_2_overview}{Experimental overview} + \subcap{fig:il15_2_overview}{Experimental overview}. Longitudinal measurements of \subcap{fig:il15_2_fc}{fold change} and \subcap{fig:il15_2_viability}{viability} for blocked and unblocked conditions expanded with \glspl{dms}. \subcap{fig:il15_2_mem}{Flow cytometry markers for \gls{dms}-expanded T - cells at day 14 for blocked and unblocked groups.}. + cells at day 14 for blocked and unblocked groups.} } \label{fig:il15_2} \end{figure*} We next tried blocking soluble \gls{il15} itself using either a \gls{mab} or an -\gls{igg} isotype control. \anti{\gls{il15}} or \gls{igg} isotype control was +\gls{igg} isotype control. Anti-\gls{il15} or \gls{igg} isotype control was added at \SI{5}{\ug\per\ml}, which according to \cref{fig:doe_luminex} was in excess of the \gls{il15} concentration seen in past experiments by over -\num{20000} times. Similarly, we observed no difference between fold change, +\num{20000} times. Similarly, there was no difference between fold change, viability, or marker histograms between any of these markers, showing that blocking \gls{il15} led to no difference in growth or phenotype. -% RESULT this can probably be worded more specifically in terms of the cis/trans -% action of IL15 In summary, this data did not support the hypothesis that the \gls{dms} platform gains its advantages via the \gls{il15} pathway. \section{Discussion} -This work provides insight for how the \gls{dms} operates and may be optimized -further. The data showing increased \pthp{} when \glspl{dms} are added and the -reverse when removed is consistent with other data we produced via \gls{doe} -showing that higher \gls{dms} concentrations lead to higher \pthp{} -(\cref{fig:doe_responses_cd4,fig:add_rem_cd4}). The difference in this case is -that we showed that altering activation signal analogously affects the \pthp{} -in the dimension of time as well as space. A similar trend was observed with -memory T cells in this aim. Our previous \gls{doe} data showed that, to a point, -lower \gls{dms} concentration leads to higher \ptmemp{} +This work provides insight for how the \gls{dms} platform operates and how it +may be optimized further. The data showing increased \pthp{} when \glspl{dms} +are added and the reverse when removed is consistent with other data we produced +via \gls{doe} showing that higher \gls{dms} concentrations lead to higher +\pthp{} (\cref{fig:doe_responses_cd4,fig:add_rem_cd4}). The difference in this +case is that altering activation signal analogously affects the \pthp{} in the +dimension of time as well as space. A similar trend was observed with memory T +cells in this aim. Our previous \gls{doe} data showed that, to a point, lower +\gls{dms} concentration leads to higher \ptmemp{} (\cref{fig:doe_responses_mem}). In this aim, we showed that decreasing activation signal temporally by removing \glspl{dms} leads to the same effect in -the \gls{tcm}, \gls{tscm} and `transitory' \gls{tscm} populations, (all of which -are included in the \ptmem{} phenotype). Taken together, these imply that -temporally or spatially altering the \gls{dms} concentration, and thus the -activation signal, has similar effects. +the \gls{tcm}, \gls{tscm} and ``transitory'' \gls{tscm} populations, (all of +which are included in the \ptmem{} phenotype). Taken together, these imply that +temporally or spatially decreasing the \gls{dms} concentration, and thus the +activation signal, increases memory and lowers CD4+ fractions. -% BACKGROUND this sounds like background? -% There are several plausible explanations for the observed phenotypic differences -% between beads and DMSs. First, the DMSs are composed of a collagen derivative -% (gelatin); collagen has been shown to costimulate activated T cells via -% \gls{a2b1} and \gls{a2b2}, leading to enhanced proliferation, increased -% \gls{ifng} production, and upregulated CD25 (IL2R$\upalpha$) surface -% expression8,10,11,41,42. +While we did not find support for our hypothesis that \glspl{dms} signal via the +\gls{a2b1} and/or \gls{a2b2} receptors, we can speculate that either the +experiment failed to block the targeted pathways or that this mechanism is +simply not relevant for our system. -While we did not find support for our hypothesis that the \gls{dms} signal -through the \gls{a2b1} and/or \gls{a2b2} receptors, we can speculate as to why -either this experiment failed and may be done better in the future, or why these -receptors may simply be irrelevant for our system. +On the first point, we did not verify that these \glspl{mab} actually blocked +their target receptors (although they were from a reputable manufacturer, \bl). +However, other groups have shown that these particular clones work at the +concentrations we used\cite{MirandaCarus2005}. Furthermore, we can safely rule +out the possibility that the \glspl{mab} never reached their targets, as they +were added immediately after the T cells were resuspended as required for cell +counting, hence their resting clustered state was disrupted. Therefore, the most +likely failure mode was that the \glspl{mab} we obtained were somehow defective +in their intended purpose, which we could experimentally verify using adhesion +assays. -On the first point, we did not verify that these \glspl{mab} indeed blocked the -receptor we were targeting. There has been evidence from other groups that these -particular clones work at the concentrations we used\cite{MirandaCarus2005}. -This does not necessarily mean that the \glspl{mab} we obtained were functional -in blocking their intended targets (although they were from a reputable -manufacturer, \bl). Furthermore, we can safely rule out the possibility that the -\glspl{mab} never reached their targets, as they were added immediately after -the T cells were resuspended as required for cell counting, hence their resting -clustered state was disrupted. - -On the second point, the collagen domains may not even be relevant to our system -depending on the nature of the \gls{stp} coating. We intended by design for the -system to be fully coated or nearly fully-coated with \gls{stp} -(\cref{fig:stp_coating}). Thus the domains that \gls{a2b1} and \gls{a2b2} may be -targeting could be sterically hindered by a layer of \gls{stp}, and if not that, -also a layer of CD3/CD28 \glspl{mab}. The other possibility is that these -domains are simply denatured to beyond recognition due to the fabrication -process for the microcarriers we used (which involves a proprietary -cross-linking step to make the material autoclave-safe). Either of these could -be tested and verified by staining the \glspl{dms} with a fluorescently-tagged -\gls{mab} and verifying binding via confocal microscopy or indirect protein -quantification as we do for the \gls{qc} of the \gls{dms}. If this test came -back negative, we would be fairly confident that the \gls{a2b1} and \gls{a2b1} -domains are either unreachable or unrecognizable. Even if it turned out that -collagen binding domains are irrelevant in the \gls{dms} system, previous -studies show that these domains can enhance proliferation and survival, and thus -adding them along with with the \glspl{mab} could enhance T cell -expansion\cite{Aoudjit2000, Gendron2003, Boisvert2007}. +On the second point, collagen domains may not even be relevant to our system +depending on the extent of \gls{stp} coating. We intended by design for the +system to be fully coated with \gls{stp} (\cref{fig:stp_coating}). Thus the +domains that \gls{a2b1} and \gls{a2b2} may be targeting could be sterically +hindered by a layer of \gls{stp}, and if not that, also a layer of CD3/CD28 +\glspl{mab}. The other possibility is that these domains are simply denatured to +beyond recognition due to the fabrication process for the microcarriers (which +involves a proprietary cross-linking step to make the material autoclave-safe). +Either of these could be tested and verified by staining the \glspl{dms} with a +fluorescently-tagged \gls{mab} and verifying binding via confocal microscopy or +indirect protein quantification as we do for the \gls{qc} of the \gls{dms}. If +this test came back negative, we would be fairly confident that the \gls{a2b1} +and \gls{a2b1} domains are either unreachable or unrecognizable. Even if it +turned out that collagen binding domains are non-existent in the \gls{dms} +system, previous studies have shown that these domains can enhance proliferation +and survival, and thus adding them along with with the \glspl{mab} could enhance +T cell expansion\cite{Aoudjit2000, Gendron2003, Boisvert2007}. We also failed to uphold our hypothesis that the \gls{dms} system gains its advantage via \gls{il15} signaling. There could be multiple reasons for why @@ -4108,7 +4096,7 @@ memory phenotypes\cite{Lodolce1998,Kennedy2000}. Second, in the case of the receptor it could be that that \glspl{mab} we purchased did not actually block, which also seems unlikely given that this clone has been observed to inhibit proliferation in the past (although like the integrin blocking experiments we -did not verify that it blocked ourselves), albeit of resting T +did not verify for ourselves that it blocked), albeit of resting T cells\cite{MirandaCarus2005}. Third, it could be that turnover of the receptor was so high that there were not enough \glspl{mab} to block (the key difference between our experiment and that of \cite{MirandaCarus2005} was that they used @@ -4116,9 +4104,9 @@ resting T cells, which are not expressing protein to nearly as high of a degree). The way to test this would be to simply titrate increasing concentrations of \gls{mab} (which we did not do in our case because the \gls{mab} was already very expensive in the concentrations employed for our -experiment). Fourth, the blocking the soluble protein may not have worked -because the \il{15} may have been secreted and immediately captured via -\il{15R$\upalpha$} either by the cell that secreted it or by a neighboring cell. +experiment). Fourth, blocking the soluble protein may not have worked because +\il{15} may have been secreted and immediately captured via \il{15R$\upalpha$} +either by the cell that secreted it or by a neighboring cell. Regardless of whether or not \il{15} is important for the overall mechanism that differentiates the \glspl{dms} from the beads, adding \il{15} or its receptor