*Purpose: We have evaluated recovery of T and B lymphocytes and their subsets after depletion by anti-thymocyte globulin (ATG) and an anti-CD20mAb (Rituximab), followed by genetically-engineered pig kidney Tx in baboons receiving an anti-CD40mAb-based maintenance immunosuppressive regimen.

*Methods: Life-supporting pig kidney Tx was performed (n=15). Induction therapy was with ATG (10mg/kg) on pre-Tx day-3, and anti-CD20mAb (10mg/kg) on day-2, aimed to achieve a CD3⁺T cell count of <500cells/mm² and complete depletion of B lymphocytes in the blood. Maintenance therapy consisted of (i) anti-CD40mAb (50mg/kg), (ii) rapamycin (trough level 6-10ng/ml), and (iii) methylprednisolone (0.125mg/kg). Follow-up was for 6 months (n=4), or until graft loss from rejection (n=7), or discontinuation for other complications (n=4). Recipient baboon blood was collected pre-Tx (day-7) and at intervals post-Tx to measure (by flow cytometry) CD3⁺T, CD4⁺T, CD8⁺T, and CD22⁺B cell numbers. In addition, CD4⁺T and CD8⁺T cell subsets (naïve, central memory [CM], effector memory [EM]) and CD22⁺B cell subsets (naïve, non-switched memory, switched memory, double-negative) were measured.

*Results: After induction therapy, the total lymphocyte count fell by 85% within 2 weeks, and subsequently recovered to only approximately 20% of baseline level after 2 months (p<0.001). The absolute numbers of CD3⁺, CD4⁺, and CD8⁺T cells fell by >90% by day 0 (day of kidney Tx) (all p<0.001), and no CD22⁺B cells were measurable in the blood after day 7 (p<0.001). T cell numbers began to recover within 2 weeks, but only to <33% of the pre-ATG baseline number throughout 6 months’ follow-up (p<0.001 compared with baseline), even if rejection developed. Virtually no CD22⁺ B cells were measured in the blood until approximately 2 months after Tx (day 60), and recovery remained at <33% of the baseline (p<0.001). At 6 months, recovery of CD4⁺ naïve (<30%), CM (<40%), and EM (<10%) cells, and of CD8⁺ naïve (<20%), CM (<20%) cells was similar (all p<0.001 vs baseline), but CD8⁺ EM cells increased to 60% of baseline. Recovery at 6 months of CD22⁺ naïve (>60%), non-switched memory (10%), switched memory (<2%), and double-negative (<10%) cells indicated that a much greater percentage were naïve in contrast to baseline.

*Conclusions: ATG and anti-CD20mAb effectively decreased the numbers of T and B lymphocytes and, in the presence of anti-CD40mAb therapy, recovery of these cells remained at approximately 33% of baseline. Although the predominant subsets of CD4⁺T and CD22⁺B cells were naïve, the predominant subset of CD8⁺T cells was EM. Importantly, we could find no correlation between T or B or subset cell recovery and the development of rejection in the graft.

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