Presented at:
ASHII 2006 Annual Meeting
San Diego, CA, October 17, 2006

Tolerogenicity of graft infiltrating lymphocytes in donor stem cell infused kidney transplant recipients treated with alemtuzumab

Mathew J, Cirocco R, Rosen A, Vallone T, Gomez C, Blomberg B, Fuller L, Burke GW, Ciancio G, Esquenazi V, Miller J

University of Miami and the VA Medical Center, Miami, FL

Aim: Absence or presence of donor reactive cells in the peripheral circulation may not be a true reflection of the in vivo immune status of the recipient. Donor reactive cells may be sequestered in the secondary lymphoid organs or even the allograft itself. The aim of the present study was to monitor the immune status within the graft.

Methods: We obtained needle biopsies of kidney allografts from two non-rejecting recipients infused with donor hematopoietic stem cells (DHSC) and treated with Campath-1H. The graft infiltrating lymphocytes were grown in a cytokine rich medium. (No cells grew out from the biopsy of a third patient, possibly indicating the absence of graft infiltrating cells.) The cells that grew in the biopsy cultures were phenotypically characterized by flow cytometric analysis. They were also functionally tested as responders in Mixed Lymphocyte Reaction (MLR), micro-Cell Mediated Lympholysis (m-CML), Enzyme-Linked ImmunoSpot (ELISpot) assay and as modulators (regulators) in MLR.

Scope:  Flow cytometric analysis revealed that the cells that grew out of the biopsies by 3 weeks in culture were of recipient phenotype distributed into CD3+CD4+ T cells and CD16/56+ NK cells [Table 1]. The cultures had significant proportions of CD4+CD25+ cells that co-expressed the regulatory receptor GITR and Fox-P3 transcripts. When the biopsy cultures were tested functionally, the following observations were made. (1) They proliferated in response to the donor stimulators in MLR giving stimulation indices ranging from 2.5 to 6.6. (2) However, in micro-CML assays they did not have cytotoxic activity against the donor targets. (3) In ELISpot assays the frequencies of IFN-γ producing cells were 15,000 – 34,700 per million; but they did not have appreciable granzyme-B producing cells, only 1,500 – 1,800 per million cells. (4) More, importantly, when tested as third component modulators in MLR cultures of recipients pre-transplant responder PBL, they strongly inhibited the anti-donor as well as anti-third party responses in a dose dependent manner. However, because of the paucity of cells, the cell subsets that functioned as immune regulators in the cultures could not be identified.

Conclusions:  Taken together these results clearly indicate that kidney allografts of DHSC infused and Campath-1H treated recipients have infiltrating cells that function as regulators of the recipients immune responses. Speculatively, these cells may facilitate allograft acceptance.

Table 1: Flow cytometric profile of cells that grew out of a biopsy (% of total cells gated on lymphocyte-monocyte populations)

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