Detection of donor-derived MHC class II^high cells.

<p>Three colour flow cytometry was used to detect highly expressing MHC class II donor cells of BMT recipients. Mononuclear cells were pre-gated concerning scatter properties. (<b>A</b>) Leukocytes expressing high density of MHC class II antigens were gated using anti-CD45 mAb (OX-1) and anti-MHC class II mAb (OX-6). (<b>B</b>) Anti-RT1B/D^u mAb (1H1A) was used to detect cells of donor origin (1H1A⁺). Data from a representative chimeric recipient treated with 6 Gy and Srl for 28 days are shown.</p

Application of Allogeneic Bone Marrow Cells in View of Residual Alloreactivity: Sirolimus but Not Cyclosporine Evolves Tolerogenic Properties

<div><p>Background</p><p>Application of bone marrow cells (BMC) is a promising strategy for tolerance induction, but usually requires strong depletion of the host immune system. This study evaluates the ability of immunosuppressants to evolve tolerogenic properties of BMC in view of residual alloreactivity.</p><p>Methods</p><p>The rat model used a major histocompatibility complex (MHC) class II disparate bone marrow transplantation (BMT) setting (LEW.1AR1 (RT1^auu) → LEW.1AR2 (RT1^aau)). Heart grafts (LEW.1WR1 (RT1^uua)) were disparate for the complete MHC to recipients and for MHC class I to BMC donors. Limited conditioning was performed by total body irradiation of 6 Gy. Cyclosporine (CsA) or Sirolimus (Srl) were administered for 14 or 28 days. Transplantation of heart grafts (HTx) was performed at day 16 or at day 100 after BMT. Chimerism and changes in the T cell pool were detected by flow cytometry.</p><p>Results</p><p>Mixed chimeras accepted HTx indefinitely, although the composition of the regenerated T cell pool was not changed to a basically donor MHC class II haplotype. Non-chimeric animals rejected HTx spontaneously. BMC recipients, who received HTx during T cell recovery at day 16, accepted HTx only after pre-treatment with Srl, although chimerism was lost. CsA pre-treatment led to accelerated HTx rejection as did isolated application of BMC.</p><p>Conclusion</p><p>Srl evolves tolerogenic properties of allogeneic BMC to achieve indefinite acceptance of partly MHC disparate HTx despite residual alloreactivity and in particular loss of chimerism.</p></div

Effects of 6 Gy of TBI on T cell pool.

<p>Three male LEW.1AR2 rats were irradiated with 6 Gy of TBI. Analysis was performed on day 3 after TBI. Control animals of identical strain, gender and age did not receive any treatment (ctrl). After counting vital mononuclear cells per μl blood as well as per spleen, analyses were performed by flow cytometry. T cells with α/β TCR expression (R73⁺) were divided into CD8⁺ and CD8^-. Propidium-iodid counter-staining was used to exclude avital cells. (<b>A</b>) Mean values of absolute numbers of α/β TCR+ cells in peripheral blood. (<b>B</b>) Absolute numbers of vital α/β TCR+ cells in spleen subdivided according to CD8 expression as mean values.</p

Courses of donor chimerism after MHC class II disparate BMT in recipients conditioned by 6 Gy of TBI and treated with immunosuppression.

<p>LEW.1AR2 male rats were irradiated with 6 Gy of TBI and received 1 x 10⁸ BMC from LEW.1AR1 donor rats one day after TBI. Recipients were additionally treated either with sirolimus (Srl) or cyclosporine A (CsA). Control animals did not receive any immunosuppression (no IS). Donor chimerism was detected within blood B cells by flow cytometry using donor-specific anti-RT1B/D^u mAb (1H1A) on days 14, 30, 60 and 100 post BMT. (<b>A</b>) Mean values of chimerism from groups receiving Srl for 14 days (Srl 14d; n = 13) or CsA for 14 days (CsA 14d; n = 11) as well as control animals (no IS; n = 11). (<b>B</b>) Mean values of chimerism from groups receiving Srl for 28 days (Srl 28d; n = 15) or CsA for 28 days (CsA 28d; n = 10). The statistical significance was appointed at * p < 0.05 for BMT groups receiving immunosuppression for 14 days and TBI alone (A) as well as for both groups receiving immunosuppression for 28 (B).</p

CD4⁺/CD4^- T cell ratio in peripheral blood of T cell regenerated BMT recipients at day 100.

<p>Peripheral blood of T cell regenerated BMT recipients at day 100 was analyzed by flow cytometry (n = 5 per group). Naïve rats of LEW.1AR1 donor and LEW.1AR2 recipient strain with same gender and similar age were analyzed as controls (n = 5 per group). Anti-α/β TCR mAb and anti-CD4 mAb were used. Mean values of CD4⁺/CD4^- ratios in α/β TCR⁺ cells are given. The type of chimerism is indicated (X = stable chimerism, no X = no persistent chimerism). Irradiation dosages and immunosuppressants (sirolimus: Srl; cyclosporine: CsA) including duration of application in days are given. The statistical significance was appointed at * p < 0.05 for naïve LEW.1AR1 as well as X 10 Gy group versus naïve LEW.1AR2 and all other groups.</p

Transplantation of BM with or without depletion of donor-derived α/β TCR⁺ cells.

<p>Transplantation of BM with or without depletion of donor-derived α/β TCR⁺ cells.</p

MHC (RT1) and CD45 (RT7) immunogenetics of strain combinations used for the bone marrow transplantation model.

<p>MHC (RT1) and CD45 (RT7) immunogenetics of strain combinations used for the bone marrow transplantation model.</p

Weight indices of recipients receiving BM with or without depletion of donor-derived α/β TCR⁺ cells.

<p><i>LEW</i>.<i>1W (RT1</i>^<i>u</i>, <i>RT7</i>^<i>a</i><i>) recipients were conditioned by 15 mg/kg of anti-RT7</i>^<i>a</i><i>mAb 3 days prior to transplantation of 1 x 10</i>^<i>8</i><i>BMC from MHC syngeneic (LEW</i>.<i>1U-7B</i>: <i>RT1</i>^<i>u</i>, <i>RT7</i>^<i>b</i><i>)</i>, <i>MHC haploidentical (LEW</i>.<i>1U-7B x LEW</i>.<i>7B</i>: <i>RT1</i>^<i>u/l</i>, <i>RT7</i>^<i>b</i><i>) or MHC disparate (LEW</i>.<i>7B</i>: <i>RT1</i>^<i>l</i>, <i>RT7</i>^<i>a</i><i>) donors</i>. <i>BM grafts were depleted from α/β TCR</i>^<i>+</i><i>cells in vitro (TCD) or were kept untreated (no TCD)</i>. <i>Thus</i>, <i>1 x 10</i>^<i>8</i><i>BMC contained 5</i>.<i>4 x 10</i>^<i>6</i><i>α/β TCR</i>^<i>+</i><i>cells (MHC syngeneic)</i>, <i>5</i>.<i>1 x 10</i>^<i>6</i><i>α/β TCR</i>^<i>+</i><i>(MHC haploidentical) and 4</i>.<i>2 x 10</i>^<i>6</i><i>α/β TCR</i>^<i>+</i><i>cells (MHC disparate)</i>, <i>respectively</i>, <i>when untreated</i>. <i>Weight indices were calculated by dividing the actual weight of the recipients through the baseline weight at the time point of BMT conditioning (= anti-RT7</i>^<i>a</i><i>mAb injection)</i>. <i>Weight indices were calculated and depicted as mean values +/- standard deviation per group (</i>^<i>…</i><i>○</i>^<i>…</i><i>depletion of</i> α/β TCR⁺ cells; –<i>■– no depletion of</i> α/β TCR⁺ cells) <i>Statistical analyses were performed applying the unpaired t test (* p < 0</i>.<i>05)</i>.</p

Biodistribution and effects of biotinylated anti-RT7^a mAb in lympho-hematopoietic compartments of RT7^a/RT7^b chimeras.

<p><i>To enable reliable flowcytometric gating of lymphocyte subsets, which will be strongly depleted by the anti-RT7^a mAb, six mixed chimeras carrying disparity only in the RT7 (CD45) antigen were generated by sublethal TBI of 7 Gy. Reconstituted rats showed a multi-lineage RT7^a-chimerism of 10 to 30 percent differing between lineages and compartments (day 0 = before mAb application). The biotinylated anti-RT7^a mAb was applied once (2 mg/kg) before the tissue compartments were analysed by flow cytometry at days 3, 7, 14, 21 and 35. The fraction of persisting RT7^a positive cells is given as percentage of all CD45⁺ cells per lineage (column height). The black column gives the fraction of RT7^a positive cells that is coated by the biotinylated anti-RT7^a mAb. T-lymphocytes (α/βTCR⁺) and NK cells (NKR-P1⁺) were strongly and compartment independently depleted, whereas B-lymphocytes (CD45RA⁺) were coated by anti-RT7^a mAb, but were not significantly reduced in cell number despite high-graded coating. (PB–peripheral blood, THY–thymus, LN–lymph node, SP–spleen, BM–bone marrow).</i></p

Effects of anti-RT7^a mAb on bone marrow level.

<p><i>A single dosage of 10 mg/kg of anti-RT7^a was injected into LEW.1W rats (n = 4). (A) BMC were analysed 7 days after mAb injection, whereby BMC were categorized by CD45 expression density and granularity using flow cytometry. Myelopoiesis showed a right shift, whereby SSC^low/CD45^low early progenitors as well as SSC^low/CD45^high lymphoid progenitors were markedly reduced. (B) Histology verified the right shift in myelopoiesis.</i></p