Earinus Wesmael, 1837 are transferred to a new genus, Chilearinus Sharkey gen. nov. Presently three Nearctic species of Earinus are recognized, i.e., Earinuserythropoda Cameron, 1887, Earinuslimitaris Say,1835, and Earinuszeirapherae Walley, 1935, and these are retained in Earinus. Earinuschubuquensis Berta, 2000 and Earinusscitus Enderlein, 1920 are transferred to Chilearinus, i.e., C.chubuquensis, and C.scitus, comb. nov. One other species is transferred to Chilearinus, i.e., Microgasterrubricollis Spinola, 1851, Chilearinusrubricollis, comb. nov. Two other Neotropical species, Earinushubrechtae Braet, 2002 and Earinusbourguignoni Braet, 2002 were described under the genus Earinus but are here transferred to Lytopylus, L.hubrechtae, and L.bourguignonicomb. nov. Two new species of Chilearinus are described, C.covidchronos and C.janbertspp. nov. The status of Agathislaevithorax Spinola,1851, Agathisrubricata Spinola,1851, and Agathisareolata Spinola, 1851 is discussed. A neotype is designated for Earinuslimitaris  and diagnosed with a COI barcode. Earinusaustinbakeri and Earinuswalleyispp. nov. are described. The status of both Earinus and Chilearinus in the Americas is discussed. A revised key to the genera of Agathidinae of the Americas is presented.The Neotropical members formerly included in  Earinus Wesmael, 1837 are transferred to a new genus, Chilearinus Sharkey gen. nov. Presently three Nearctic species of Earinus are recognized, i.e., Earinuserythropoda Cameron, 1887, Earinuslimitaris Say,1835, and Earinuszeirapherae Walley, 1935, and these are retained in Earinus. Earinuschubuquensis Berta, 2000 and Earinusscitus Enderlein, 1920 are transferred to Chilearinus, i.e., C.chubuquensis and C.scitus, comb. nov. One other species is transferred to Chilearinus, i.e., Microgasterrubricollis Spinola, 1851, Chilearinusrubricollis, comb. nov. Two other Neotropical species, Earinushubrechtae Braet, 2002, and Earinusbourguignoni Braet, 2002 were described under the genus Earinus but are here transferred to Lytopylus, L.hubrechtae, and L.bourguignoni comb. nov. Two new species of Chilearinus are described, C.covidchronos and C.janbert spp. nov. The status of Agathislaevithorax Spinola,1851, Agathisrubricata Spinola,1851, and Agathisareolata Spinola, 1851 is discussed. A neotype is designated for Earinuslimitaris  and diagnosed with a COI barcode. Earinusaustinbakeri and Earinuswalleyi spp. nov. are described. The status of both Earinus and Chilearinus in the Americas is discussed. A revised key to the genera of Agathidinae of the Americas is presented.Neotropical species formerly included in c oxidase subunit I (COI) gene using standard insect primers LepF1 (5′-ATTCAACCAATCATAAAGATATTGG-3′) and LepR1 (5′-TAAACTTCTGGATGTCCAAAAAATCA-3′) (Molecular work was carried out at the CBG using standard protocols. A leg from each frozen-then-oven-dried specimen was destructively sampled for DNA extraction using a glass fiber protocol . ExtractATCA-3′) . If inithttp://www.boldsystems.org/index.php/IDS_OpenIdEngine); (2) paste the COI sequence of the query organism (in forward orientation) into the query box and search against the appropriate library ; (3) the search results page shows the top hits based on percentage similarity starting with the closest matches ; (4) use the Tree-Based Identification button to generate a neighbor-joining tree and find the query taxon (name in red). This allows you to visualize how distant the query sequence is from the closest matches.The BOLD database can be used to identify specimens using the following steps: (1) navigate to the identification tab of the BOLD Systems database  can also be employed to differentiate them. Members of Lytopylus differ most significantly in that they lack vein Cub in the hind wing. See couplet 25 in the key below.Notauli absent; hind coxal cavities open; tarsal claws with basal lobes; second submarginal cell quadrate, never petiolate; foretibia lacking sclerotized spines/pegs; hind wing Cub strong and emanating from an angle on the basal cell. Most similar morphologically to Head. Lateral carina on frons (as found in members of Alabagrus) absent; interantennal space slightly raised above antennal sockets; gena not extended ventroposteriorly into sharp prominence; mandible dorsoventrally flattened (twisted); labial palpus with 4 segments, third segment slightly more than ½ length of apical segment. Mesosoma. Propleuron lacking a sharp bump; notauli absent; mesoscutum smooth with a median pit (presumably a remnant of notauli), postscutellar depression absent; propodeum mostly smooth, sometimes with weak smooth sculpture medially; sclerite between hind coxal cavities and metasomal foramen absent. Precoxal groove absent or smooth and weakly impressed. Legs. Foretibia lacking dull pegs (unlike Earinus); mid- and hind tibia with blunt apical or preapical pegs; all tarsal claws with a rounded basal lobe. Wings. Forewing RS+Ma vein mostly present but not usually completely tubular; second submarginal cell large, quadrate and usually  higher than long; RS of forewing complete to wing margin; hind wing r and r-m cross veins absent; hind wing vein Cub strong and emanating from an angle on the basal cell. Metasoma. First median tergite smooth, longer than apical width, lateral longitudinal carina absent or weak and short; remaining terga smooth; ovipositor ranging from as long as the body to twice the length of the body, but this is based on small sample of a few dozen species.Unknown.This is a species-rich genus with hundreds of species, based on specimens identified by MS. It is widespread in Chile and southern Argentina. A few species are found at high altitudes as far north as Ecuador and Colombia.Chilearinus in a broader concept of Earinus. Agathidinae from Chile. Since members of Chilearinus are by far the most species-rich of Chilean agathidines, and since his descriptions do not contradict membership in the genus, these species are probably members of Chilearinus, i.e., Agathislaevithorax, Agathisrubricata, and Agathisareolata. They certainly are not members of Agathis since this genus does not extend into the southern regions of South America. These specimens should be in the Hymenoptera collection of Maximilian Spinola whose collection is housed in the Museo Regionale di Scienze Naturali (MRSN) in Turin (Torino). One of us (MS) could not locate these specimens during a visit to MSRN in 1985, but a specimen of Chilearinus, Microgasterrubricollis Spinola, 1851, was present. Microgaster may seem an odd place for placement of what we now consider an agathidine, but such was the classification at the time. It is clear from the following that Spinola knew the species was closely related to Earinus, “Este Microgastro habria pertenecido á [sic] la primera seccion del G. Microdus, N. V. Es., y al sub-género Earinus Wesm.” .♀, Chile, Región IX, PN Nahualbuta, COI barcode. BOLD sample ID H1145. BOLD BIN code BOLD:AAV0870. GenBank Accession Code OL702761.AATTTTATATTTTATATTTGGAATTTGATCGGGAATTTTAGGTTTATCAATAAGTTTAATTATTCGAATAGAATTAAGAGTAGGGGGTAATTTTATTGGTAATGATCAAATTTATAATAGAATTGTNGCTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGAGGATTTGGAAATTGATTAATTCCATTAATATTGGGGGGGCCAGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCATTATTATTATTAATTTTAAGGTCTTTAATTAATGTTGGGGTAGGTACTGGATGAACTGTTTATCCTCCTTTATCATTAAATATAAGTCATAGTGGTATATCTGTAGATTTAGCTATTTTTTCTTTACATATTGCTGGAATTTCTTCAATTATAGGTGCTATAAATTTTATTACAACTATTTTAAATATGTGAATAATTAATATTAAAATTGATAAAATACCTTTATTAGTTTGATCAATTTTAATTACGGCAATTTTATTATTATTATCTTTGCCAGTTTTAGCTGGAGCTATTACTATATTATTAACAGATCGTAATTTAAATACTAGATTTTTTGATCCTTCTGGAGGAGGAGATCCAATTTTATATCAACATTTATTTSee key.None.Named in acknowledgment of the covid pandemic occurring during the production of this manuscript.Taxon classificationAnimaliaHymenopteraBraconidaeSharkeysp. nov.AB9C892C-E5CA-5F6A-BE83-F0ED72EAEB87http://zoobank.org/AF4C4A3B-EBD8-4305-AF39-DC9176C868A837.493°S, 72.582°W, 1168 m, 8.ii.2005, Heraty, .♀, Chile, Región IX, PN Nahualbuta, COI barcode. BOLD sample ID H12114. BOLD BIN: BOLD:AEM7846. GenBank Accession Code OL702760.TTTTAGGATTATCAATAAGTTTAATTATTCGAATAGAATTAAGAGTAGGTGGTAATTTTATTGGTAATGATCAAATTTATAATAGGATTGTNACTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGAGGATTTGGAAATTGATTAATTCCATTAATATTAGGGGGTCCAGATATAGCCTTCCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCATTATTATTATTAATTTTAAGATCTTTAATTAATGTTGGAGTAGGTACTGGATGAACTGTTTATCCTCCTTTATCATTAAATATAAGTCATAGTGGTATATCTGTAGATTTGGCTATTTTTTCTTTACATATTGCTGGAATTTCTTCAATTATAGGGGCTATAAATTTTATTACAACTATTTTAAATATATGAATAATTAATATTAAAATTGATAAAATACCTTTATTAGTTTGATCAATTTTGATTACAGCAATTTTATTATTATTATCTTTACCAGTTTTAGCTGGGGCTATTACTATATTATTAACAGATCGTAATTTAAATACTAGATTTTTTGATCCTTCTGGAGGGGGAGATCCAATTTTATATCAACATTTATTTTGATTTTTSee key.None.A conjunction of Paul Hebert and Dan Janzen in recognition of their enormous contributions towards the conservation of nature.Taxon classificationAnimaliaHymenopteraBraconidaeWesmael, 18373EB02370-41B3-561B-B842-5117B904EE03Earinus, i.e., E.erythropoda Cameron, 1887, E.limitaris , and E.zeirapherae Walley, 1935, and here we describe two more, Earinusaustinbakeri sp. nov. and Earinuswalleyi sp. nov. In the Nearctic, Earinus is common and widespread with the southernmost record being the sole recognized specimen of E.erythropoda from northern Sonora state, Mexico. Earinus differs from Chilearinus in the possession of pegs/spines in the foretibia and the characters given in the key.In the Americas, there are three previously recognized species of Hymenoptera Institute  and borrowed specimens, there are probably between eight and 12 species in the Nearctic region. They are extremely similar in color, but there are obvious differences among specimens in body dimensions, degree of punctation, color of the hind coxae, ocellar configuration, ovipositor length, length and density of setae on the ovipositor sheath, and dimensions of the first metasomal tergum. Unfortunately, these are not sufficient to allow confident delineation of species limits. For example, the differences in the key between E.limitaris and E.erythropoda are trivial. There are numerous specimens scattered over the Nearctic region that will key to E.erythropoda, but they might all be E.limitaris, or the two nominal species may be conspecific, or there may be multiple cryptic species. Likewise, there are probably a number of undescribed Nearctic species that will key to either E.zeirapherae or E.austinbakeri. In other words, the key is sufficient to discriminate among the barcoded species and E.zeirapherae but not among these and the undescribed species. The key is presented in part to satisfy the code of Zoological Nomenclature to act as a diagnosis for E.austinbakeri and E.walleyi. Only dense sampling of COI barcodes and perhaps other genes will supply the information necessary to delimit Nearctic Earinus species.Based on the collection in the Taxon classificationAnimaliaHymenopteraBraconidaeSharkeysp. nov.107889AA-0F66-5D06-A568-CA774826DA26http://zoobank.org/D169A981-8A48-4E53-B1D1-CB072D89814744.2829°N, 77.7963°W, 131 m, 05–20.Jun.2014 . BOLD sample ID BIOUG33065-A05, BOLD BIN code BOLD:ADL5164. GenBank Accession Code OM158425.♀, Canada, Ontario, Ferris Provincial Park, Consensus barcode based on four specimens.ATTTTATATTTTATATTTGGGATTTGATCYGGAATTGTGGGKTTATCAATAAGTTTAATTATTCGTATGGARTTAAGAGTAGGGGGBAATTTAATTGGKAATGATCAAATTTATAATAGTATTGTTACTGCTCATGCATTTATTATAATTTTTTTTATAGTTATRCCAATTATAATTGGTGGGTTTGGTAATTGGTTAATTCCTTTAATATTAGGRGGTCCCGATATRGCTTTCCCTCGAATGAAYAATATAAGRTTTTGATTATTAATTCCTTCTTTATTATTATTAATTTTAAGATCTTTAATTAATATTGGGGTTGGAACTGGTTGAACGGTYTATCCTCCTTTATCATTRAATATAAGTCATAGTGGTATATCTGTTGATTTGGCTATTTTYTCTTTACATATTGCGGGRATTTCTTCTATTATAGGGGCAATAAATTTTATTACTACTATTTTAAATATATGAATAATAAATATTAAAGTTGATAAAATGTCTTTATTRATTTGATCAATTTTAATTACTGCTATTTTATTATTATTATCTTTACCTGTTTTAGCRGGRGCAATTACTATATTATTAACAGATCGTAATTTAAATACAAGATTTTTTGATCCTTCTGGAGGTGGGGATCCAATTTTATATCAACATTTATTTE.austinbakeri but differing by the characters given in the key as well as having the ovipositor sheath more setose. The COI barcodes of the two species differ by 6.29% (p-distance), reinforcing the conclusion that they are different species.Very similar to http://www.boldsystems.org).BIOUG01028-C01, BIOUG01028-F12, BIOUG32793-A05. These are sample IDs; the data for these specimens can be found by searching for these codes on BOLD .E.limitaris, or several more species may have similar morphologies. COI barcode data are needed. Several line drawings, modified from The sole identified specimen is the holotype. It differs little from many specimens that are widespread in the United States. It could be that they all belong to Taxon classificationAnimaliaHymenopteraBraconidaeEAE38190-F29E-5D79-A78E-22DCB293E7E9Bassuslimitaris Say, 1835.38°55'N, 78°49'W, 30.viii–19.ix.2005 . BOLD sample ID H1141. BOLD BIN code BOLD:AAU8493. GenBank Accession Code OM237775.♂, USA, West Virginia, Hardy County, 3 mi. NE Mathias, COI barcode based on 9 specimens.Consensus AATTTTATATTTTATATTTGGAATTTGATCAGGAATTTTAGGTTTATCAATAAGATTAATTATTCGAATAGAATTAAGDATAGGTGGTAATTTRATTGGTAATGATCAAATTTATAATAGTGTTGTTYCTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATGATTGGRGGRTTTGGRAATTGATTAGTTCCTTTAATATTGGGRGGTCCTGATATAGCTTTYCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCTTTATTATTATTAATTTTGAGTTCTTTAATTAATATTGGGGTRGGGACTGGKTGAACAGTTTATCCTCCRTTATCTTTAAATATAAGRCATAGTGGAATATCAGTTGATTTAGCTATTTTTTCATTACATATYGCAGGAATTTCTTCAATTATAGGGGCAATAAATTTTATTACTACTATYATAAATATATGAATAATAAATATTAAAATTGATAAAATACCTTTATTAGTTTGATCAATTTTAATTACTGCTATTTTATTATTATTATCATTRCCAGTTTTAGCTGGRGCAATTACTATATTATTAACAGATCGAAATTTRAATACAAGATTTTTTGATCCTTCTGGAGGGGGGGATCCAATTTTATATCAACATTTATTTSee key.http://www.boldsystems.org).ASGLE-0444, ASGLE-0446, ASGLE-0449, ASGLE-0451, ASGLE-0452, ASGLE-0445, BIOUG01022-D11, BIOUG32892-B07. These are sample IDs; data on them can be found by searching for these codes on BOLD  which differs by only 2.54% (p-distance) from E.limitaris , as well as either Missouri or Indiana, or both. It is unknown if Taxon classificationAnimaliaHymenopteraBraconidaeSharkeysp. nov.8DA974A7-E3F6-5E6F-A2B2-FF42AC77EBAAhttp://zoobank.org/BDFBEADA-2082-46A5-B648-EB181E09CBB558.3734°N, 94.1342°W, 3–7.vii.2007, Malaise trap . BOLD sample ID. 07PROBE-20853, BOLD BIN code BOLD:AAF9894. GenBank Accession Code FJ413805.♀, Canada, Manitoba, Churchill pump house, 15 km S Churchill, Goose Creek Road, Consensus barcode based on four specimens.TATTTTATATTTTATATTTGGAATTTGATCAGGTATTGTAGGTTTATCAATAAGATTAATTATTCGAATGGAATTAAGAGTGGGRGGTAATTTAATTGGRAATGATCAAATTTATAATAGTATTGTTACTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGGGGRTTTGGTAATTGATTARTCCCATTAATATTGGGAGGTCCTGATATAGCTTTCCCTCGTATAAATAATATGAGATTTTGATTATTAATCCCYTCTTTATTAATATTAATTTTAAGATCTTTAATTAATATTGGAGTAGGGACTGGTTGGACAGTTTATCCTCCKTTATCATTAAATATAAGTCATAGTGGAATATCTGTTGATTTGGCTATTTTTTCTTTACATATTGCGGGRGTTTCTTCTATTATAGGGGCAATAAATTTTATTACTACTATTTTAAATATRTGAATAATAAATATTAAAATTGATAAAATGTCTTTATTAATTTGATCAATTTTAATTACTGCTATTTTATTATTATTRTCTTTACCAGTTTTAGCAGGAGCTATTACTATATTATTAACAGATCGTAATTTAAATACAAGATTTTTTGATCCTTCYGGAGGGGGTGACCCAATTTTATATCAACATTTATTTE.zeirapherae, differing by the characters given in the key as well as having the ovipositor sheath less setose. The COI barcodes of the two species differ by 6.29% (p-distance) all but ensuring that they are different species.Very similar to http://www.boldsystems.org).All are from the same locality as the holotype, 07PROBE-23096, 07PROBE-23097, 09PROBE-A0304. These are specimen IDs; more data on the specimens can be found by searching for these codes on BOLD , former research scientist at the Canadian National Collection and author of Taxon classificationAnimaliaHymenopteraBraconidaeWalley, 1935766B1C04-99FA-52A3-8733-9263AE5C92E1♀, Grand River, Nova Scotia, 11.May.1932 (M. L. Prebble) No. 3847 .Tortricidae: Aclerishudsoniana, Choristoneurarosaceana, Rhyacioniaadana, Zeirapheracanadensis, Zeirapheragriseana, and Zeirapheraratzeburgiana. Since there are many species, including E.austinbakeri and E.walleyi, that are morphologically similar to E.zeirapherae, all hosts that do not belong to the genus Zeiraphera need confirmation.The following are all reported as hosts by Zeirapheraratzburgiana. Contrary to the image of the holotype in Figure The holotype Fig.  is from (Modified from


However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum (ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm3, forming abundant contacts with other organelles4. Dysregulation of endoplasmic reticulum morphology is tightly linked to neurologic disorders and cancer6. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180–microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions The endoplasmic reticulum proteins CLIMP63, kinectin and p180 bind preferentially to subsets of microtubules with different post-translational modifications, thereby linking the ‘tubulin code’ to the intracellular distribution of membrane organelles. Although this code has been implicated in cargo selection and directed organelle movement2, how it is decoded to mediate transport and control distribution remains largely unknown.Eukaryotes compartmentalize cellular functions within distinct organelles, and regulation of organelle position is critical for cell health. Organelles are transported bidirectionally by motor and adaptor proteins along microtubules7. The ER is a compelling candidate for exploiting the complexity of the tubulin code, since it spreads throughout the cytoplasm in association with microtubules and makes abundant organelle contacts10. Most studies of ER shaping and organelle contacts have emphasized peripheral tubular ER. How the denser perinuclear ER is shaped and asymmetrically distributed remains largely unknown, although three ER membrane-bound proteins—CLIMP63, p180 and KTN1—localize prominently to perinuclear ER and are considered sheet-forming proteins11. Even so, depletion of CLIMP63 may paradoxically lead to the expansion of ER matrices or sheets in the periphery12, and perinuclear ER matrices or sheets remain abundant even upon simultaneous knockdown of all three proteins, prefiguring more complex functional roles11.Endoplasmic reticulum (ER) comprises structurally and functionally divergent membrane compartments that include interconnected tubules, perinuclear matrices and sheets, and the nuclear envelope12, peripheral ER in CLIMP63-knockout cells is populated with increased numbers of dense matrices or sheets—a ‘dispersed’ phenotype. KTN1 knockout also disperses ER, whereas p180-knockout cells exhibit a contrasting ‘clustered’ ER phenotype, with the peripheral network remaining tubular and perinuclear ER collapsing asymmetrically into a smaller area at one side of the nucleus  to visualize their microtubule associations in cells ) that shape the tubules. The polygonal network is generated via atlastin-mediated tethering and fusion of tubules at three-way junctions and distributed via cytoskeletal interactions2. Modifications are dynamic and rapidly reversible, but evidence for how they affect microtubule-related functions has been limited2. We have shown here that KTN1 preferentially binds perinuclear polyglutamylated microtubules with long glutamate chains, whereas p180 binds glutamylated microtubules with either short or long chains. By contrast, CLIMP63 has a higher threshold for response to microtubule glutamylation. We cannot exclude that increased affinity at higher glutamate numbers for TTLL7-modified microtubules stems from additional chains that TTLL7 initiates on tubulin tails, and not only from introduction of longer chains. Conversely, p180 is more sensitive to any increase in glutamate numbers on the tubulin tail and robustly binds both mono- and polyglutamylated microtubules. This differential effect on microtubule binding according to glutamylation state has previously been observed for the microtubule-severing ATPase spastin29 and may represent a general feature of this modification, enabling fine tuning of molecular interactions. Thus, a small difference in the number of glutamates added to tubulin side chains may exert a substantial qualitative effect on ER distribution. Other ER-localized, microtubule-binding proteins30 are likely to contribute to overall cellular ER positioning. Indeed, even in p180 and KTN1 double-knockout cells, TTLL7 overexpression still disperses ER, suggesting the involvement of other ER proteins. Moreover, tubular ER selectively moves along acetylated microtubules27, further indicating that ER distribution is broadly sensitive to microtubule modifications.Microtubule diversity can be achieved via different tubulin gene products, differential interactions with microtubule-associated proteins and numerous post-translational modifications31, and axonal microtubules are highly glutamylated2. Thus, p180 may affect microtubule remodelling by differentially recognizing glutamylated axonal microtubules. Of note, although dysregulation of ER shaping and microtubule polyglutamylation lead to different neurodegenerative diseases33, these diseases share some similar cellular phenotypes, including mitochondrial distribution defects and axon degeneration, suggesting possible convergence.The ability of cells to dynamically control ER distribution through differential microtubule modifications has important functional implications. For instance, p180 regulates microtubule remodelling in axons1, and their ability to selectively distribute organelles relies on a tubulin code. Our results indicate that ER distribution is mediated via specific membrane-bound proteins with differential binding to different levels and types of microtubule glutamylation, broadly affecting distributions of most other organelles. ER thus interprets the tubulin code to regulate movement and positioning of cellular organelles. Rather than imbuing each organelle with its own sensing and response mechanisms, cells achieve organizational efficiency by using ER as a first-line sensor and responder. This role is exemplified during nutrient starvation, when cells increase CLIMP63 protein levels to move ER towards the perinuclear region, which also clusters lysosomes for efficient autophagic degradation. Then, cells harness enhanced p180–microtubule binding to redistribute ER and lysosomes for a proper reset. There are likely to be other ER proteins that also decipher the tubulin code, with important implications for ER function in health and disease.When ER positioning is disrupted, distributions of other organelles are affected. Microtubules have key roles in organelle distribution21. pCMV6_p180s-myc-Flag (RC218816), pCMV6_KTN1-myc-Flag (RC219832), pCMV6_TTLL4-myc-Flag (RC205206) and pCMV6_CCP1-myc-Flag (RC220826) were obtained from Origene Technologies. pcDNA3.1_TTLL6-Flag (OHu07095), pcDNA3.1_CCP5-Flag (Ohu28493), pcDNA3.1_CCP6-Flag (OHu24335) and pcDNA3.1_p180L (OHu24745) were obtained from GenScript. Mutants of CLIMP63, p180s and KTN1 were generated in a pcDNA3.1(+) vector with a C-terminal HA-epitope tag. Specifically, Ser-to-Glu mutants  of CLIMP63 were synthesized by GenScript. pcNDA3.1-KTN1, pN1-KTN1-mApple, pC1-sp-mScarlet-KTN1, pN1-KTN1-mNeonGreen, 2×Strep-p180s_29-381-mNeonGreen, 2×Strep-p180s_29-381-mNeonGreen and CLIMP63-mNeonGreen-2×Strep were also constructed using standard cloning procedures. 5′ and 3′ UTR of KTN1 were synthesized by Integrated DNA Technologies and ligated into the pN1-KTN1-mNeonGreen vector. Human TTLL4, TTLL6, TTLL7, CCP1, CCP5 and CCP6 inserts were also cloned into pCMV14-3×Flag and pN1-mApple. DNA oligonucleotides were synthesized by Integrated DNA Technologies.GFP-mCherry-LC3 was a gift from Juan S. Bonifacino. mEmerald-Sec61β, pcDNA3.1_CLIMP63-HA, mApple-SiT (Golgi apparatus), mKO-SKL (peroxisome), YFP-KDEL (ER) and CFP-LAMP1 (lysosome) were constructed as described previously34. Camptothecin (S1288), DC661 (S8808), etoposide (S1225) and MG132 (S2619) were purchased from Selleckchem. LysoSensor Green DND 189 (L7535), G418 (11811098) and puromycin (A1113802) were from Thermo Fisher Scientific. Centrinone B  was from Tocris. GTP (G8877), ATP, Taxol (T7402), tunicamycin (SML1287), Earle’s Balanced Salts  and Duolink In Situ PLA kit  were from Sigma-Aldrich. The Cathepsin L Activity Assay Kit (Fluorometric) (ab65306) was obtained from Abcam.LD540 was provided by C. Thiele2. HEK 293T cells were transfected with Avalanche-Everyday Transfection Reagent . U2OS and COS7 cells were electroporated using Cell Line Nucleofector Kit V  and Cell Line Nucleofector Kit R  following the manufacturer’s instructions. Amounts of key plasmids transfected are (per 1 × 106 U2OS cells or 5 × 105 COS7 cells): 0.3 μg CLIMP63-HA, 0.5 μg p180s-HA, 2 μg KTN1-mApple, 1 μg CLIMP63-mEmerald (for overexpression), 2 μg p180s-mEmerald, 4 μg p180L-mEmerald, 4 μg KTN1-mEmerald, as well as (in various vectors) 1 μg TTLL4, 1 μg TTLL6, 0.3 μg TTLL7, 4 μg CCP1, 0.5 μg CCP5, and 1 μg CCP6.All cell lines were obtained from the American Type Culture Collection: HEK 293T (CRL-11268), COS7 (CRL-1651), HeLa (CCL-2), RPE1 (CRL-4000) and U2OS (HTB-96) cells. HEK 293T, COS7 and HeLa cells were cultured in Dulbecco’s Modified Eagle Medium , RPE1 cells were cultured in DMEM/F12 (1:1) Medium , and U2OS cells were cultured in McCoy’s 5A medium (Thermo Fisher Scientific16600108); all were supplemented with 10% fetal bovine serum  and 1×penicillin/streptomycin/amphotericin B (Thermo Fisher Scientific 15240112) at 37 °C with 5% CO6 U2OS cells using Lonza Cell Line Nucleofector Kit V.For RNAi knock down of AKAP450, two siRNAs targeting ATATGAACACAGCTTATGA and AACTTTGAAGTTAACTATCAA were synthesized by Eurofins Genomics. Cells were transfected using Avalanche-Omni Transfection Reagent  with 20 pmol siRNA for 3 days. For RNAi knockdown of CCP5, ON-TARGETplus siRNA sets targeting human CCP5 were purchased from Horizon Discovery (LQ-009468-00-0005), and 60 pmol siRNAs were transfected per 1 × 10Primary antibodies used: mouse monoclonal anti-AKAP450 , rabbit polyclonal anti-Atlastin2 , rabbit polyclonal anti-Atlastin3 , rabbit monoclonal anti-Catalase , mouse monoclonal anti-Climp63 , mouse monoclonal anti-Flag M2 , rabbit polyclonal anti-GFP , mouse monoclonal anti-GM130 , rabbit polyclonal anti-GM130 , mouse monoclonal anti-HA , rabbit polyclonal anti-kinectin , rabbit monoclonal anti-kinectin , mouse monoclonal anti-Lamp1 , rabbit polyclonal anti-LC3 , rabbit polyclonal anti-Lunapark , mouse monoclonal anti-Myc , rabbit polyclonal anti-p180 , rabbit polyclonal anti-Pericentrin , rabbit polyclonal anti-polyglutamylation (polyE) , mouse monoclonal anti-glutamylation clone GT335 , rabbit polyclonal anti-REEP2 , rabbit polyclonal anti-REEP3 , rabbitpolyclonal anti-REEP4 , rabbit polyclonal anti-REEP5 , rabbit polyclonal anti-reticulon3 , rabbit polyclonal anti-reticulon4 , rabbit polyclonal anti-RPL3 , rabbit polyclonal anti-TOM20 , mouse monoclonal anti-TOM20 , rabbit polyclonal anti-TRAPα , rat monoclonal anti-α-tubulin Alexa Fluor 647 , mouse monoclonal anti-α-tubulin , mouse monoclonal anti-β-tubulin . Alexa Fluor 405/488/568/633 conjugated goat anti-rabbit/mouse IgG (H+L) highly cross-adsorbed secondary antibodies were from Thermo Fisher Scientific. HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies were from Santa Cruz Biotechnology.7 and selected using 200–1,000 μg μl−1  G418 for two weeks; green-positive cells were sorted into mono-clones by flow cytometry using a MoFlo Astrios cell sorter (Beckman Coulter) and cultured in the presence of 200 μg μl−1 G418 for 2–3 weeks. Proliferated clones were verified by immunoblotting and fluorescence imaging.To generate U2OS cells stably expressing mEmerald-Sec61β, cells were transfected with the mEmerald-Sec61β35. The targets used were: CLIMP63:GCCGCGCCCGCCATGCCCTCGG; p180 in U2OS: GGTGTCGACTTTCTCCATGAAGG; p180 in COS7: GACACCAGGAAGATGCCAATGG; KTN1: GAAAAGCCAGAAGAAGAGG and GTTAGGGAAAGAAAAAAGAAGG.All CRISPR–Cas9 knockout assays used eSpCas9(1.1)CCAGCCCGCGGCCCGAGCCGCCGCCGCGCCCGCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGtccggactcagatctcgagctcaagcttcgaattctgcagtcgacggtaccgcgggcccgggatccCCCTCGGCCAAACAAAGGGGCTCCAAGGGCGGCCACG;For knock-in of CLIMP63, the same target as in CLIMP63 knockout was used, and a PCR fragment with 37 bp homology arms on each side of the mEmerald-coding sequence was used as a homologous recombination template as follows: GGCCTCCTCGGCTTGGCCGCCGTCGAGCCCGCCGTCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGAGCCCGCCGTCTACTTCAAGGAGCAGTTTCTGGAC. Note that amino acids 18–20 (EPA) were appended to both sides, acting as a linker.. To generate mEmerald-calreticulin knock-in COS7 cells, wild-type Cas9 with a gRNA targeting the end of the signal sequence of calreticulin (GAGCCCGCCGTCTACTTCAAGG) was selected, and a PCR fragment with 36 bp homology arms on each side of the mEmerald-coding sequence was used as a homologous recombination template as follows: 15 before further analysis.To deplete the centrosome, cells were treated with 125 μM CNB for 1 week as describedCells were quickly rinsed with PBS, directly lysed with sample buffer , and boiled for 5 min. Proteins were then resolved by SDS–PAGE using Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad Laboratories) and transferred to nitrocellulose membranes using the Trans-Blot Turbo RTA Midi Nitrocellulose Transfer Kit (Bio-Rad Laboratories) following the manufacturer’s instructions. Membranes were blocked with 4% milk in TBST , and incubated with primary antibody (diluted in blocking buffer) at 4 °C overnight. After washing with TBST, membranes were incubated with secondary antibody at room temperature for 2 h, followed by intensive washing with TBST. Immunoreactive proteins were visualized with GE Healthcare LS ECL Prime Western Blotting Detection Reagent (RPN2236) and imaged using a ChemiDoc XRS+ (Bio-Rad). Band intensities were quantified using Fiji software (NIH).−1 LD540 in PBS for 5 min. Cells were mounted using Fluoromount-G (SouthernBiotech) and imaged using a Zeiss LSM880 confocal microscope in Airyscan mode equipped with a 63 × 1.4 NA Plan-Apochromat oil objective (Carl Zeiss). Images were acquired using ZEN software (Carl Zeiss) and processed with ZEN software or Fiji (NIH).Cells were fixed with 4% paraformaldehyde in PBS (Lonza) for 30 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Alternatively, for immunostaining of glutamylation (GT335) and polyglutamylation (polyE), cells were fixed and permeabilized with cold methanol for 5 min at −20 °C. Then, after blocking with 3% BSA for 30 min, cells were immunostained with polyE antibody at 4 °C overnight, then with polyE and GT335 together at 4 °C overnight, followed by secondary antibody staining at room temperature for 1 h, and finally with anti-α-tubulin Alexa Fluor 647 at room temperature for 2.5 h. For staining of lipid droplets with LD540 dye, cells were incubated with 0.1 μg mlr,θ)-space representation of the cell’s fluorescence distribution. For analysis referring to ‘normalized’ data, we account for the shape of the cytoplasm by finding the radius at each angle where the nuclear envelope and the edge of the cell are located. The fluorescence data were then rescaled to a normalized axis with the cytoplasm between the nuclear envelope and the cell periphery scaled from 0 to 100%. The nucleoplasm is scaled to stretch between −25 and 0, as a control. .Three-dimensional images were acquired using a Zeiss LSM880 confocal microscope in Airyscan mode and reconstructed using ZEN software (Zeiss Microscopy). Summed intensity projections were generated using floating point notation to carry precision. A custom macro in Fiji-ImageJ was used to define the centre of the nucleus and remove the signal of neighbouring cells to avoid perturbing the results. From the manually defined centre, a radius was drawn out past the furthest point on the cell and swept through 360° in 0.1° steps, taking a line profile each time and rescaling the data to correct for artifacts generated by the square shape of the pixels. The resulting data represents an  for 30 min on ice. Cell lysates were centrifugated twice at 20,000g for 20 min at 4 °C. The supernatant was supplemented with 1 mM GTP and 40 μM Taxol and incubated at 4 °C or 37 °C for 30 min for tubulin polymerization before centrifugation at 20,000g for 30 min at 4 °C or 37 °C, respectively. The resulting pellets (P) and supernatants (S) were collected and subjected to immunoblot analysis. In some experiments, only the pellets and supernatants of the 37 °C samples are shown.To test the microtubule-binding affinities of CLIMP63, p180 and KTN1, cells were lysed in PIPES buffer  was performed according to the manufacturer’s instructions. Samples were observed under a Zeiss LSM880 confocal microscope with a 20 × 1.0 NA objective using the Airyscan function. The total intensity of the PLA signal per cell was quantified using Fiji software.g at 4 °C for 30 min. Supernatants were combined with Strep-Tactin XT beads (IBA Lifesciences) and rotated gently for 3 h. After extensive washing with lysis buffer (PBS plus 500 mM NaCl and 1% Triton X-100) and then wash buffer (IBA Lifesciences), bound proteins were eluted with Strep-Tactin XT Elution Buffer (IBA Lifesciences). Eluted proteins were subjected to multiple rounds of PBS dilution and concentration using 10 kDa protein concentrators (Sigma-Aldrich), before being aliquoted and frozen in liquid nitrogen.Deletion fragments of p180  and KTN1  as well as full-length CLIMP63 were expressed as fusions with mNeonGreen-2×Strep in HEK 293T cells. 48 h post-transfection, cells were lysed in PBS (Lonza) plus 500 mM NaCl, 1% Triton X-100, and protease inhibitors and then centrifugated at 30,00036. TTLL4 and TTLL6 were expressed in Escherichia coli and purified as previously described18. TTLL7 was also expressed in E. coli and purified as previously described19 . Taxol-stabilized microtubules were polymerized out of 98.5% unmodified tubulin and 1.5% biotinylated brain tubulin29 (Cytoskeleton T333P). Unmodified microtubules were modified using TTLL4, TTLL7 or TTLL6 at 1:10 molar ratio of enzyme to tubulin at room temperature in 20 mM HEPES (pH 7.0), 50 mM NaCl, 10 mM MgCl2, 1 mM glutamate, 1 mM ATP, 0.5 mM TCEP, and 10 μM Taxol for 4.5 h for TTLL4, between 20 min and 2 h for TTLL7, and between 7.5 and 22 h for TTLL6. Control microtubules were incubated with the enzymes under the same conditions but with aspartate, which is not a substrate for TTLL glutamylases, instead of glutamate. Enzymes were removed through a high-salt wash as previously described29. The extent of glutamylation was determined by liquid chromatography–electrospray mass spectrometry29 (LC–MS). The spectra display the characteristic distributions of masses with peaks separated by 129 Da, which corresponds to one glutamate . Next, a solution containing 60 mM Pipes (pH 6.8), 0.7 mM MgCl2, 0.7 mM EGTA, 50 mM KCl, 10 mM 2-mercaptoethanol, 10 μM Taxol, 1% F127 Pluronic, 1.4 mg/ml casein, 20 mM glucose, glucose oxidase, and catalase was flushed into the chamber, followed by the same solution containing 4.7 nM mNeon-labeled p180, KTN1 or CLIMP63. Images were acquired after allowing for equilibration for 5 min at room temperature using total internal reflection fluorescence (TIRF) microscopy at an exposure of 100 ms for the GFP channel. Unlabelled microtubules were visualized using interference reflection microscopy38. Multiple fields of view were imaged. Background corrected line scan average intensities were measured using Fiji software. Multiple chambers were quantified for each condition.For microtubule-binding assays, microtubules were immobilized in chambers made of silanized glassTotal mRNA were extracted using TRIzol (Thermo Fisher Scientific 15596018) and Direct-zol RNA Miniprep , then reverse-transcribed using the SuperScript IV First-Strand Synthesis System . Real-time PCR primers, designed by a free online tool developed by Integrated DNA technologies, were as follows: CCP5-RT: GACTGCCAGGAACTGCTAAA and AGGAGCTCCCGATGGTAATA; GAPDH-RT: GGTGTGAACCATGAGAAGTATGA and GAGTCCTTCCACGATACCAAAG.Real-time PCR was performed using Applied Biosystems PowerUp SYBR Green Master Mix  with Applied Biosystems QuantStudio 6 Flex real-time PCR instrument. Data were collected and analysed in QuantStudio Real-time PCR Software and Microsoft Excel using the 22. Images were acquired with a Zeiss LSM880 confocal microscope equipped with a 32-channel multi-anode spectral detector (Carl Zeiss) using a 63×/1.4 NA objective lens, at 37 °C and with 5% CO2. Fluorophores were excited simultaneously using 458, 514 and 594 nm lasers and a 458/514/594 nm beam splitter, with images collected onto a linear array of 32 photomultiplier tube elements in λ mode at 9.7 nm bins from 468 to 687 nm. Spectra were defined by imaging singly labelled cells for each of the fluorophore reporters, using the same acquisition and laser settings as for multiply labeled cells. Multispectral images were unmixed using the linear unmixing package in ZEN (Carl Zeiss).Multispectral imaging was performed as described previouslyz-projection was performed using maximum projection before quantification. The mCherry-positive vesicles indicate autophagosomes already fused with lysosomes, as the GFP signal would be quenched by the acidic environment of lysosomes; vesicles with both GFP and mCherry fluorescence indicate autophagosomes not yet fused with lysosomes. Quantifications of these two types of vesicles were performed manually using Fiji software.For autophagosome–lysosome fusion assessments, U2OS cells were transfected with GFP-mCherry-LC3 for 24 h, treated with EBSS for 2 h before fixation with 4% paraformaldeyde in PBS, and imaged using a Zeiss LSM880 confocal microscope in Airyscan mode equipped with a 63 × 1.4 NA Plan-Apochromat oil objective (Carl Zeiss). A For lysosome acidification assays, U2OS cells were labeled with 1 μM LysoSensor Green DND 189 for 4 min and immediately imaged within one minute with a Zeiss Axio microscope using a 20×/0.4 NA objective. Images were captured with ZEN software, and total intensities of each cell were quantified in Fiji.6 cells were assayed in each sample.Cathepsin L activity assays were carried out using the Abcam Cathepsin L Activity Assay kit  following the manufacturer’s instructions; 1 × 10P values are shown on top of the corresponding columns, as determined by one-way ANOVA followed by Dunnett’s multiple comparisons test, Mann–Whitney test, Kruskal–Wallis test or by unpaired two-sided t-test as indicated in the figure legends. When representative images are shown, at least three repeats were performed except for Extended Data Figs. No statistical method was used to predetermine sample size. All groups were randomly assigned and every group represents a distincttreatment or condition. Data were not analysed in a double-blinded manner. All comparisons were performed using Graphpad Prism or Microsoft Excel software. Data are expressed as means ± s.d., Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-021-04204-9.Supplementary InformationThis file contains Supplementary Text and Supplementary Figs 1–7.Reporting SummaryPeer Review FileSupplementary Video 1Wild-type or CLIMP63, p180 KO U2OS cells stably expressing mEmerald–Sec61β were labelled with Lysotracker Red, starved with EBSS, and recorded for 2.5 h with imaging at 1 min intervals. Scale bar, 10 µm.


This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses.T cell receptor (TCR) clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cell’s transcriptional program with its TCR identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques (RM), a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline, ‘RM-scTCR-Seq’, which, for the first time, enables RM specific single cell TCR amplification, reconstruction and pairing of RM TCR’s with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track  More icities) .To date, clonotype tracking technologies have been developed for both mouse and human TCRs, facilitating studies of T cell clonal dynamics at an unprecedented level of molecular sensitivity –11. HoweTo address these limitations, nonhuman primate (NHP) models (particularly using rhesus macaques (RM)) have been essential , and havThe identification and tracking of TCR clonotypes are of critical importance for each of the clinical settings described above, in order to understand the molecular mechanisms driving immune protection or disease pathogenesis. For acute GVHD (aGVHD), dissecting the link between T cell clonal architecture and disease is particularly relevant, given that donor-derived alloreactive T cells are the main culprits in the aGVHD-mediated destruction of the skin, intestine, and liver –34. Accuin vitro and in vivo identification and tracking of RM derived T cell clonotypes. Pairing of in vivo identified alloreactive clonotypes with their transcriptional profile revealed a highly activated cytotoxic CD8 T cell signature during NHP aGVHD.We now describe the design and validation of RM-specific primers compatible with the human 10x Genomics single cell sequencing platform, which accurately amplifies the RM TCR alpha and beta regions. Amplified fragments aligned to our custom-assembled and annotated RM TCR reference permit, for the first time, the This study was conducted in strict accordance with (USDA) United States Department of Agriculture regulations and the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. It was approved by the Massachusetts General Hospital and Biomere Animal Care and Use Committees. T cells were obtained from healthy colony RM or from animals who underwent allogenic HCT in the setting of previously published studies .137Cs) radiation. Responder PBMCs were stained with cell trace violet  as per manufacturer’s instructions. 2×105 T cell-enriched responder PBMCs along with an equal number of stimulator PBMCs were added to each well in a 96-well plate (Corning) in X-vivo-15 medium (BioWhitaker) supplemented with 10% FBS (Irvine Scietific) and incubated at 37°C for a total of 5 days. Cell culture media change was performed on day 3 of the culture. At the end of 5 days, cells were stained with an extracellular antibody for CD3 (clone SP34-2), CD20 (clone 2H7) and CD14  for 20min at 4°C, and high-, medium- and non-proliferating CD3+ T cells (identified based on the dilution of CTV) were sorted and processed for single cell sequencing using the Chromium Next GEM Single Cell 5’ Reagent Kit v1 with optimized RM primers as described below.RM PBMCs were isolated from whole blood by Ficoll gradient centrifugation, and then used for MLR assays either immediately, or after liquid nitrogen cryopreservation in 10% dimethyl sulfoxide (DMSO)/90% fetal bovine serum (FBS). At the time of the MLR, stimulator PBMCs were irradiated with 3500 cGy of , we also validated primers and PCR conditions compatible with the newer Next GEM Single Cell 5’ v2 Sample Index Plate TT  (see below). https://assets.ctfassets.net/an68im79xiti/31W4aZOJ8C2ipxzTrcQIWn/72c5c2bc3dd7784f44f5218ca345ce04/CG000086_ChromiumSingleCellV_D_J_ReagentKits_UG_RevM.pdf). Primer sequences are listed in Single Cell V(D)J v1: GEMs were generated using v2 Gel Beads and the v1 Target Enrichment kit substituting the custom primers for the off-the-shelf human/mouse T Cell Mix 1 and 2 premixed primers. Sample Indexing was performed using the i7 Multiplex Kit plate . TCR alpha and beta primers were designed to perform in a nested PCR approach to optimize target enrichment of the respective constant regions Table 1.PCR was performed with an initial denaturation temperature of 98°C for 45 seconds, followed by denaturation at 98°C for 20 seconds. Annealing was optimal at 67°C for 30 seconds with extension of 72°C for 1 min. A total of 20 PCR cycles were performed with a final extension step at 72°C for 1 min.https://assets.ctfassets.net/an68im79xiti/57JaTECQNBPSpyDz8oucdi/92da9f62521ea1f5c781ba234bd0a0f5/CG000331_ChromiumNextGEMSingleCell5-v2_UserGuide_RevC.pdf). TRAC inner and outer primers were 2996bp apart, and the beta chain inner and outer primers were 354bp apart. Primer sequences are listed in Next GEM Single Cell 5’ v2: GEMs were also generated using v2 reagent kits for GEM generation and library preparation while substituting the custom RM primers for the off-the-shelf human/mouse T Cell Mix 1 and 2 premixed primers. Sample Indexing was performed using the Dual Index Kit TT (10x Genomics PN-1000215). Primers Table 2 PCR was performed with an initial denaturation temperature of 98°C for 45 seconds, followed by denaturation at 98°C for 20 seconds. Annealing was optimal at 62°C for 30 seconds with extension of 72°C for 1 min. A total of 20 PCR cycles were performed with a final extension step at 72°C for 1 min. Sequencing results utilizing v2 primers are listed in The reference transcriptome Data S4 1. We first filtered Ensembl’s gtf file for v104 of the macaca mulatta gene annotation using cellranger’s mkgtf command with the following attributes:–attribute=gene_biotype:protein_coding –attribute=gene_biotype:lincRNA –attribute=gene_biotype:miRNA –attribute=gene_biotype:antisense –attribute=gene_biotype:IG_LV_gene –attribute=gene_biotype:IG_V_gene –attribute=gene_biotype:IG_V_pseudogene –attribute=gene_biotype:IG_D_gene –attribute=gene_biotype:IG_J_gene –attribute=gene_biotype:IG_J_pseudogene –attribute=gene_biotype:IG_C_gene –attribute=gene_biotype:IG_C_pseudogene –attribute=gene_biotype:TR_V_gene –attribute=gene_biotype:TR_V_pseudogene –attribute=gene_biotype:TR_D_gene –attribute=gene_biotype:TR_J_gene –attribute=gene_biotype:TR_J_pseudogene –attribute=gene_biotype:TR_C_gene2. We next edited cellranger’s mkref program to use more than one thread using the command:sed -i ‘88s/num_threads/#num_threads/’/cellranger-6.0.0/bin/rna/mkref3. We then created the reference transcriptome using cellranger’s mkref command: cellranger-6.0.0/bin/rna/mkref –genome=MMulatta_Ensembl_v104 –fasta=MMulatta_Ensembl_v104_combined.fasta –genes=MMulatta_Ensembl_v104_combined_filtered.gtf –nthreads=32 –memgb=1281. We used version 6.0.2 of Cellranger to create the initial VDJ reference based on IMGT , 36.2. We used the following command to generate the initial reference: cellranger-6.0.2/lib/bin/fetch-imgt –genome vdj_IMGT_rhesus –species “Macaca mulatta”.3. This version of the VDJ reference did not include constant regions, so we added the following sequences - obtained from IMGT and Ensembl - to the reference:>340|TRAC*01 IMGT|TRAC|C-REGION|TR|TRA|None|01ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGGCTCTAAATCCAATGACACCTCTGTCTGCCTATTTACTGATTTTGATTCTGTAATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCGATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>341|TRAC*02 TRAC_205_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|02ATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>342|TRAC*03 TRAC_201_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|03ATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>343|TRAC*04 TRAC_202_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|04ATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>344|TRAC*05 TRAC_203_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|05ACTGGGGTAAACAACCTCTTCTTTGGGACTGGAACAAGACTCACCGTTCTTCCAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGGCTCTAAATCCAATGACACCTCTGTCTGCCTATTTACTGATTTTGATTCTGTAATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>345|TRAC*06 TRAC_204_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|06ATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>346|TRBC1*01 NW_001114291|TRBC1|C-REGION|TR|TRB|None|01AGGACCTGAAAAAGGTGTTCCCACCCAAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACGCTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGGAAAGAGGTGCACAGTGGGGTCAGCACGGACCCACAGCCCCTCAAGGAGCAGCCCGCCCTCGAGGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCACAACCCCCGCAACCACTTCCGCTGCCAAGTCCAGTTCTATGGGCTCTCGGAGGATGACGAGTGGACCGAGGACAGGGACAAGCCCATCACCCAAAAGATCAGCGCCGAGGTCTGGGGTAGAGCAGACTGTGGCTTCACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTGGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGTGCCCTCATGTTGATGGCCATGGTCAGAGGAAGGATTTC>347|TRBC1*02 IMGT000073|TRBC1|C-REGION|TR|TRB|None|02AGGACCTGAAAAAGGTGTTCCCACCCAAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACGCTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGGAAAGAGGTGCACAGTGGGGTCAGCACGGACCCACAGCCCCTCAAGGAGCAGCCCGCCCTCGAGGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCAGCCACCTTCTGGCACAACCCCCGCAACCACTTCCGCTGCCAAGTCCAGTTCTATGGGCTCTCGGAGGATGACGAGTGGACCAGGACAGGGACAAGCCCATCACCCAAAAGATCAGCGCCGAGGTCTGGGGTAGAGCAGACTGTGGCTTCACCTCAGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTGGGGAAGGCCTCCCTGTATGCTGTGCTGGTCAGTGCCCTCATGTTGATGGCCATGGTCAAGAGGAAGGATTTC>348|TRBC2*03 IMGT000073|TRBC2|C-REGION|TR|TRB|None|01AGGACCTGAAAAAAGTGTTCCCACCCAAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACGCTGGTGTGCCTGGCCACAGCTTCTACCCCGACCACGTGGAGTTGAGCTGGTGGGTGAACGGGAAAGAGGTGCACAGTGGGGTCAGCACGGACCCACAGCCCCTCAAGGAGCAGCCCACCCTCGAGGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCACAACCCCCGCAACCACTTCCGCTGCCAAGTCCAGTTCTATGGGCTCTCGGAGGATGACGAGTGGACCGAGGACAGGGACAAGCCCATCACCCAAAAGATCAGCGCTGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCTGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCThis resulted in an initial set of 348 sequences. We then checked these sequences for premature stop codons, frameshifts, and other potential problems using 10x’s enclone software, v0.5.42. This identified 44 problematic sequences which were removed, leaving us with a final total of 304 VDJ segments.Samples were aligned using the Cellranger multi pipeline (v6.0.2), with VDJ libraries listed as “vdj-t” to indicate T cell libraries. Samples were aggregated with the Cellranger aggr pipeline (v6.0.2) with normalize=none. Our reference transcriptome was created with the Cellranger makeref command, and used genome assembly Mmul_10 as the reference genome , and EnsWe used FASTQC to assess the quality of our sequencing data. After alignment of this data, we loaded the filtered_feature_bc_matrix.h5 file from Cellranger multi into Seurat and removed genes which were present in less than 5 cells. This resulted in a dataset with 38,147 cells by 16,163 genes. Following the recommendations in Lucken and Theis  to normaWe then limited the dataset to samples from the PBMC, the MLR experiments, and the GVHD target organs. We then removed genes present in less than 5 cells, resulting in a dataset with 23,618 cells by 15,195 genes. This dataset was normalized using the same pipeline previously described in this section, retaining 22 PC’s from the PCA.Clonotypes used in the gene expression analysis were identified using cellranger’s default settings in “cellranger vdj” for the scTCR-Seq libraries. Clonotypes used for the Shannon Diversity metric calculations  were identified by grouping together T cells on the basis of the CDR3 region in the alpha chain or beta chain. This is to provide a more stringent test of the specificity of clonotype calling, as Cellranger’s default settings do not allow for clonotypes to be shared across different donors. The Morisita index was calculated using the “vegan” package in R.As discussed in the Results section, the clonotypes used for the alpha chain and beta chain Morisita heatmaps . The primers targeting the beta chain were specifically designed to amplify both TRBC1 and TRBC2. Primer length, CG content and annealing temperatures were adjusted to be used with the forward primers supplied in the human 5’ 10x Genomics kit. Cycling conditions were modified from the original 10x protocol to allow optimal amplification of the targeted region in RM 2. We haRM-scTCR-Seq primers annealed to the alpha and beta RM constant region and amplified transcripts covering the complete alpha and beta loci including the variable V and J regions for the alpha as well as V, D and J regions for the beta locus.  and can We assessed the quality of our primers using summary statistics from 10x’s ‘Cellranger’ software , and found that our primers consistently amplify high-quality reads from the TCR alpha and beta chains. For this assessment, we prepared peripheral blood mononuclear cells (PBMCs) from three representative RMs  and used these PBMCs to perform RM-scTCR-Seq. We observed that 49-52% of the aligned reads mapped to the alpha chain and 22-25% of the reads aligned to the beta chain Table S2In addition to demonstrating that the sequencing reads mapped to known TCR loci, we were also able to reconstruct the alpha and beta chains from these reads Table S2These data demonstrate, for the first time, efficient amplification of the RM TCR alpha and beta region by utilizing optimized RM primer pairs compatible with the human 5’ 10x Genomics single cell sequencing platform. Amplified reads enabled high quality beta chain reconstruction in almost all, and reconstruction of full TCRs  in a significant number of sequenced cells.in vitro assays or in vivo disease models enables the evaluation of the clonal repertoire, clonal dynamics, and the tracking of specific T cells over time. To validate our single cell TCR sequencing and analysis pipeline, we first utilized an in vitro allo-proliferation assay: the mixed lymphocyte reaction (MLR) (Identification of T cell clonotypes using on (MLR) –47. To pon (MLR)  (Table on (MLR) .Using the RM-scTCR-Seq pipeline, we were also able to track individual clonotypes throughout the three MLR peaks  (To further assess our ability to accurately track T cell clonotypes samples) , 50 betw). Importantly we were also able to identify clonotypes that existed in both the Blood sample and the high-proliferating population  or beta chain MI (MI <0.001), demonstrate the specificity of the RM-scTCR-Seq approach  or not (termed `MLR-`). Each MLR+ cluster incorporated at least 37 cells Table 3.ted gene , 52 was d VISION  to scored VISION  as well d VISION .in vivo utility of the RM-scTCR-Seq pipeline.These results demonstrate the feasibility of combining RM-scTCR-Seq and scRNA-Seq to identify and interrogate alloreactive T cell clonotypes in aGVHD target organs in RM, providing proof-of-concept of the TCR repertoire analysis has become a fundamental tool to understand the immune responses to infections and vaccines, as well as the immunopathogenesis of rejection after solid organ transplantation, and GVHD after HCT , 54–58. Prior to the work described herein, two major limitations significantly hindered the development of T cell clonotype tracking in RM. They were (1): The lack of optimized, single cell sequencing-compatible primers covering both the TCR alpha and beta region for RM; (2) The poor genomic annotation of the alpha and beta loci in RM, which resulted in inadequate reconstruction of amplified VDJ PCR reads. To address both of these technical barriers, we have designed a robust set of RM-specific TCR primer pairs which anneal to the constant region of the alpha and beta TCR loci, and which are compatible with the commonly used single cell sequencing platform from 10x Genomics. Optimizing PCR cycling and temperature conditions resulted in efficient amplification of the alpha and beta regions of the RM TCR and generated productive TCR reconstructions. Additionally, we created an updated reference for the TCR alpha and beta chain region, enhancing our ability to annotate the assembled RM TCR’s.www.10xgenomics.com/resourses/dataset). The difference in the alignment rate may be explained by differing levels of annotation for these organisms: 10x provides human references which have been refined over time, but this resource does not exist for RM, which necessitated our creation of a new reference (see Methods) by combining elements of the Ensembl and IMGT references, and then carefully filtering out sequences identified by 10x’s enclone software that contained errors . As annotation of the RM TCR improves, we anticipate some gains in read mapping. As mentioned earlier, we have noticed that UTR regions are annotated in the human and mouse VDJ references, and we expect that addition of these sequences to the RM reference will improve alignment rates. We additionally tested whether our reads adequately covered all genomic V(D)J regions and confirmed that in the 30 primary VDJ samples analyzed, reads mapped to 91% (179 of 196) of all V(D)J regions from our reference J gene. We note that this mapping efficiency is somewhat less than the ~91% of reads mapping to the human VDJ reference, in a human dataset provided by 10x  and the National Cancer Center. VT is supported by an ASTCT New Investigator Award and the CIBMTR/Be The Match Foundation Amy Strelzer Manasevit Research Program Award. AS is supported by the Searle Scholars Program, the Beckman Young Investigator Program, a Sloan Fellowship in Chemistry, and the NIH . LK is supported by NIH grants U19 Al1051731, R01 HL095791, P01 HL158504, and by a Leukemia and Lymphoma Society TRP grant.AS reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Hovione, Third Rock Ventures, Ochre Bio, FL82, and Dahlia Biosciences unrelated to this work. AS has received research support from Merck, Novartis, Leo Pharma, Janssen, the Bill and Melinda Gates Foundation, the Moore Foundation, the Pew-Stewart Trust, Foundation MIT, the Chan Zuckerberg Initiative, Novo Nordisk and the FDA unrelated to this work. LK is on the scientific advisory board for HiFiBio and Mammoth Biosciences. She reports research funding from Kymab Limited, Magenta Therapeutics, BlueBird Bio, and Regeneron Pharmaceuticals. She reports consulting fees from Equillium, FortySeven Inc, Novartis Inc, EMD Serono, Gillead Sciences, Vertex Pharmaceuticals, and Takeda Pharmaceuticals. LK reports grants and personal fees from Bristol Myers Squibb that are managed under an agreement with Harvard Medical School.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.