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Adaptive immunity is dispensable for salamander appendage regeneration

Chimezie H. Umeano, Marlene J. OesterleiD, Ameneh GhaffariniaiD, Ugonna Okeke, Natalie Larsson, Sapna MeenaiD, Emma BolechiD, Camila Rosat ConsiglioiD, Elif EroğluiD, András SimoniD, Nicholas D. LeighiD

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Abstract

Complex multi-tissue regeneration capacity varies across vertebrates. Mammals are amongst the least regenerative species, while salamanders can regenerate complex tissues such as limbs and tails throughout life. Previous studies have shown that innate and adaptive immune cells are present during salamander limb regeneration. While innate immune cells have been shown to promote limb regeneration, it is unknown whether adaptive immunity is responsive to amputation or plays a role in appendage regeneration. Here we show that during limb regeneration in axolotls, the immune response is characterized by a coordinated immunoregulatory signature including the downregulation of antigen presentation, cytokine secretion, and T cell activation. We corroborate this transcriptomic data in vivo using skin allografts in newts and define the blastema as an immunosuppressed niche. To test the role of adaptive immune cells in regeneration, we generated Recombination activating gene 1 deficient (Rag1-/-) newts. Rag1-/- newts lack antigen receptor recombination and show a marked reduction of adaptive immune cells. We find that Rag1-/- newts do not reject allografts, confirming their functional immunodeficiency. Finally, we demonstrate that both larval and adult newts regenerate appendages in the absence of adaptive immunity. Our work demonstrates that the adaptive arm of the immune system is not required for appendage regeneration and establishes an important model for novel experimental approaches in comparative immunology and regenerative biology.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (8 Sept 2026) · CC BY 4.0

Rag1-knockout newts lacking adaptive immunity regenerate larval limbs, tails and adult limbs without overt gross differences from controls; newt blastema tolerates allografts, and axolotl regenerating limbs show immunosuppressive transcriptional shifts.

Earlier work linked amphibian appendage regeneration to adaptive immune responses only correlatively (changes in circulating lymphocytes, effects of immunosuppressants, retarded regeneration during an ongoing allograft response). This study tests the question genetically with Rag1-deficient newts and provides an immunodeficient salamander line.

Key results

  1. ShownIn reprocessed axolotl limb scRNA-seq, immune cells were abundant, and T cells, B cells and myeloid cells shifted away from pro-inflammatory programs in regenerating limbs versus non-regenerating controls.

    About 44% of captured cells were leukocytes during wound healing. T cells downregulated Stat4, Ifngr1 and Ets1; myeloid cells downregulated antigen presentation and cytokine/interferon response genes, already by 72 hours post amputation. Controls were pooled intact and contralateral limbs. · Limb amputation and subsequent regeneration…

  2. SuggestedThe newt blastema tolerates skin allografts, whereas the same grafts on a non-regenerating hindlimb are rejected, which supports the blastema as an immunosuppressed niche.

    GFP-positive donor tissue was still detectable in 7 of 11 animals (63%) at 11 weeks after grafting into the blastema, versus 0/4 control hindlimb grafts. · The blastema niche is immunosuppressed

  3. ShownTwo Rag1 frameshift newt lines (1 bp and 7 bp deletions) lack immunoglobulin V(D)J recombination, have few splenic T cells and cannot reject allografts, so they are functionally immunodeficient.

    No immunoglobulin 5' RACE product in Rag1-/- spleen versus WT. CD3+ cells per spleen section were reduced (Cohen's d = 2.56) and spleens were smaller (d = 2.39). WT and heterozygous newts rejected grafts at 38 to 52 days post-transplantation; Rag1-/- grafts persisted to 365 days (n = 3 per group). · Rag1 deficiency abolishes V(D)J recombination in…; Rag1-/- newts have markedly reduced T cells; Rag1-/- newts are immunodeficient as evidenced…

  4. SuggestedRag1-/- newts regenerate limbs and tails as larvae and limbs as adults, with blastema formation and outgrowth comparable to controls by morphology and timing.

    Limb n = 5 Rag1-/- (pre-metamorphic); tail n = 2 Rag1-/-. In post-metamorphic limbs, time-to-stage comparisons were not significant (p = 0.69 early bud, 0.26 medium bud, 0.11 palette, 0.11 digit differentiation; Rag1-/- n = 8). · Pre-metamorphic P. waltl appendage regeneration…; Adaptive immunity is dispensable for adult newt…; Figure legends

  5. SuggestedStrong Rag1 disruption is associated with reduced survival, likely due to immunodeficiency; homozygous mutants are under-represented among adults.

    Median survival of highly edited F0 crispants was 376 days versus 460 days for lowly edited animals (p = 5 x 10^-4). Homozygous mutants were 9 of 28 larvae but 2 of 32 adults (chi-square 33.29, p < 0.0001). F2 survival over about 200 days did not differ significantly. · Rag1-/- leads to impaired survival

Methods, data and reproducibility

Reprocessed published axolotl limb regeneration scRNA-seq to profile immune cell states, then used skin allografting in Pleurodeles waltl newts and CRISPR/Cas9 Rag1 knockout lines to test immunosuppression of the blastema and the need for adaptive immunity in regeneration.

  • Axolotl limb regeneration scRNA-seq time course, reprocessed from Leigh et al. 2018 (intact, contralateral and regenerating limbs) · axolotl · SRP167700
  • Skin allografts into blastema versus hindlimb controls (GFP donor) · Pleurodeles waltl (newt) · n = 11 blastema grafts, 4 controls
  • Rag1-/- newt lines (F2), spleen immunostaining, survival and genotype ratios, allograft rejection · Pleurodeles waltl (newt) · n = CD3: Rag1-/- 3, Rag1+/- 3, WT 1; genotyped 28 larvae, 32 adults
  • Limb and tail amputation and regeneration scoring in pre- and post-metamorphic newts · Pleurodeles waltl (newt) · n = Post-metamorphic limbs: Rag1-/- n = 8, Rag1+/- n = 6, Rag1+/+ n = 5
Methods & tools
Seurat, CellBender, CyteTypeR, edgeR, clusterProfiler, EggNog, FlashFry, CRISPR/Cas9 RNP injection, ICE-Synthego, TIDE, IgBLAST, 5' RACE-PCR
Reproducibility gaps
Availability of the Rag1 newt lines and the GFP transgenic donor line is not stated; newt experiments have no deposited raw data.
Limitations (5)
  • The study cannot exclude compensation by innate or innate-like lymphoid cells in place of adaptive immunity. · stated by the authors
  • Analyses focused on gross regenerative outcomes and may miss subtle defects in patterning, cellular composition or tissue function. · stated by the authors
  • Treg requirements seen in other species are not ruled out in animals with a full adaptive lymphocyte compartment, and other regeneration paradigms such as heart were not tested. · stated by the authors
  • Group sizes are small for several readouts (Rag1-/- tail regeneration n = 2, spleen CD3 staining n = 3 per genotype, one WT animal), and regeneration was mainly scored by morphology and stage timing. · noted in this summary
  • The transcriptomic immune profiling is in axolotl and reprocessed from earlier data, while functional tests are in newts; early bud stage had only two replicates, precluding statistics. · noted in this summary

Read this if you work on regeneration or comparative immunology and want genetic evidence on whether adaptive immunity is needed for salamander appendage regeneration, or want an immunodeficient newt model.

Citations
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