Skewed HSC Clonal Output of T-Cells in an Aged Rhesus Macaque after CD3e-Immunotoxin Treatment

Background:

Lymphodepletion is often required for cancer therapy, organ transplantation, and the treatment of severe immunological diseases. People of advanced age (>55 years old) are at a higher risk for such diseases and show significantly increased morbidity and mortality with delayed and/or incomplete immune recovery following cytotoxic therapies. Our understanding of hematopoietic homeostasis and why the risks of lymphodepleting treatments are exacerbated in the elderly remains incomplete. Recent retroviral tagging studies, including our own, have shown that hematopoietic homeostasis is maintained through complex yet well-orchestrated polyclonal repopulation by heterogeneous HSC and progenitors, but clonal repopulation under stress conditions (such as lymphodepletion) has not yet been well characterized. Here we demonstrate hematopoietic stem cell (HSC) clonal behaviors prior to and after CD3e-immunotoxin treatment in one of our nonhuman primates (animal 95E132) that we have followed for up to 18 years (equivalent to approximately 60-70 human years) post-transplant.

Methods:

After 16 years of follow-up after HSC transplant, animal 95E132 was treated with 8 doses (25 ug/kg/dose) of CD3e-immunotoxin (CD3e-IT: monoclonal antibodies against CD3e-conjugated with Diphtheria toxin). Hematopoietic recovery was monitored for an additional 19 months by a complete blood cell count, flow cytometry, T-cell receptor V beta (TCRvb) spectratyping, and quantitative vector integration site (VIS) sequencing (Kim et al, Journal of Virology, 2010; Suryavanshi et al, JoVE, 2017). HSC clonal profiles in T-cell, B-cell, Granulocytes, Monocytes, and NK cells in the peripheral blood were analyzed over time. Clonal profiles in lymph nodes, spleen, bone marrow, thymus, and gut tissues were analyzed after necropsy. Two treatment-naïve animals (RA1209 and RA1174) were also treated with 8 doses (25 ug/kg/dose) of CD3e-IT to assess the effects of CD3e-IT on various blood lineages, including T-cell, B-cell, Granulocytes, Monocytes, and NK Cells, in different organs. Peripheral blood, bone marrow, and lymph nodes were collected at both one month before and 3 days after the CD3e-IT. Other lymphoid and non-lymphoid organs were collected after necropsy.

Results:

CD3e-IT treatment in the control animals, RA1209 and RA1174, showed effective T-cell depletion in the peripheral blood and bone marrow. The levels of T-cell depletion in different lymph nodes and other test organs varied notably. After CD3e-IT treatment in animal 95E132, the T-cell fraction rebounded to its normal level in 2 months, with T-cell recovery was achieved by repopulation of a few dominant clones. Such clonal dominance was maintained in peripheral T-cells until necropsy (19 months post-immunotoxin). HSC clonal profiles of T-cells after CD3e-IT shared similar patterns with the clonal profiles in bone marrow and spleen. Interestingly, the clonal profiles of thymus and lymph node T-cells were similar to those of pre-treatment blood T-cells and granulocytes (Pearson's r value of 0.75-0.91). The TCRvb repertoire was severely skewed after CD3e-IT. The recovery of naïve T-cells was slow but measurable at 19 months post-IT. B-cell, monocytes, and NK cells showed significant clonal fluctuations over time, whereas granulocytes remained highly stable ( r > 0.90).

Conclusion:

Our data suggest that peripheral expansion of a subset of T-cell clones is the first means by which T-cells repopulate following CD3e-IT treatment in aged primates. Thymic contribution occurs, but is slow and limited. T-cell repopulation in some lymphoid organs differs from that in blood and may indicate segregated homeostatic expansion. Those T-cell clones that repopulate and dominate are a very small subset of all T-cell clones, but it is unclear whether the fates of the dominant clones are predetermined. Despite the systemic and severe perturbation occurred in the T-cell pool, there was no notable sign of oligoclonal repopulation or skewed clonality in other blood lineages.