Viability of PB and CB naïve and memory T cells after treatment with CAMPATH.

<p><b>A, C, E, and G</b> show the percentage of apoptotic cells in resting/activated naïve CD4, naïve CD8, memory CD4, and memory CD8 T cells respectively. <b>B, D, F, and H</b> show the percentage of necrosis in resting/activated naïve CD4, naïve CD8, memory CD4, and memory CD8 T cells respectively. *represents p value<0.05, **represents p value<0.005, ***represents p value<0.005. Significant differences were observed between resting and activated naïve and memory T cells across all fresh samples with p value<0.005 (n = 5).</p

CD52 expression and effects of CAMPATH on HSC and HSPC.

<p><b>A and B</b> show the level of CD52 expression in CD45^high/low lymphoid and myeloid progenitors in cultures of CB and PB HSC respectively. <b>C and D</b> show the number of colony from CB SC and mPB SC respectively at day 14. <b>E and F</b> show the percentage of NK cells formed during the 35 days of culture of CB SC and mPB SC respectively. *represents p value<0.05, **represents p value<0.005, ***represents p value<0.005 (n = 3).</p

Viability of PB and CB Treg cells after treatment with CAMPATH.

<p><b>A</b> shows the percentage of apoptotic cells in resting/activated Treg cells. <b>B</b> shows the percentage of necrosis in resting/activated Treg cells. *represents p value<0.05, **represents p value<0.002, ***represents p value<0.005. Significant differences were observed between resting and activated Treg cells across all fresh samples with p value<0.005 (n = 5).</p

CD52 expression on different CB and PB cell subsets.

<p><b>A and C</b> show the level of CD52 expression in fresh and frozen resting immune cells respectively. <b>B and D</b> show CD52 expression in fresh and frozen activated immune cells respectively. *represents p value<0.05, **represents p value<0.005, ***represents p value<0.005 (n = 5).</p

Viability of activated PB and CB immune cells after treatment with CAMPATH-1H in the presence or absence of complement.

<p><b>A, C, E, G, and I</b> show the percentage of apoptotic cells in naïve T cells, memory T cells, Treg cells, NK cells, and NKT cells respectively. <b>B, D, F, H, and J</b> show the percentage of necrosis in naïve T cells, memory T cells, Treg cells, NK cells, and NKT cells respectively. *represents p value<0.05, **represents p value<0.005, ***represents p value<0.005 (n = 4/5).</p

The Effects of CAMPATH-1H on Cell Viability Do Not Correlate to the CD52 Density on the Cell Surface

<div><p>Graft versus host disease (GvHD) is one of the main complications after hematological stem cell transplantation (HSCT). CAMPATH-1H is used in the pre-transplant conditioning regimen to effectively reduce GvHD by targeting CD52 antigens on T cells resulting in their depletion. Information regarding CD52 expression and the effects of CAMPATH-1H on immune cells is scant and limited to peripheral blood (PB) T and B cells. To date, the effects of CAMPATH-1H on cord blood (CB) cells has not been studied. Here we aimed to analyze CD52 expression and the effects of CAMPATH-1H on fresh or frozen, resting or activated, PB mononuclear cells (PBMC) and CB mononuclear cells (CBMC). In resting state, CD52 expression was higher in CB than PB T cell subsets (653.66±26.68 vs 453.32±19.2) and B cells (622.2±20.65 vs 612.0±9.101) except for natural killer (NK) cells where CD52 levels were higher in PB (421.0±9.857) than CB (334.3±9.559). In contrast, CD52 levels were comparable across all cell types after activation. CAMPATH-1H depleted resting cells more effectively than activated cells with approximately 80–95% of apoptosis observed with low levels of necrosis. There was no direct correlation between cell surface CD52 density and depleting effects of CAMPATH-1H. In addition, no difference in cell viability was noted when different concentrations of CAMPATH-1H were used. CD52 was not expressed on HSC but began to be expressed as the cells differentiate, implying that CAMPATH-1H could potentially affect HSC differentiation and proliferation. Our study provides insightful information, which contributes to the better understanding in the use of CAMPATH-1H as part of the conditioning regime in HSCT.</p></div

T cell reconstitution in patients after cell therapy.

<p>P1, a child with Fanconi anaemia, underwent a second mismatched donor, CD34 selected stem cell graft after in the context of relapsed MDS. Donor HSVTK/CD34 modified T cells were infused in two dose aliquots and were detectable at low level in peripheral blood for over 12 weeks before the patient died of disease relapse. The persistence of non-modified T cells reflects the reduced intensity conditioning and absence of serotherapy. P2, an infant with RAG1 deficient SCID had no pre-existing T cell immunity and was conditioned whist infected with H1N1 influenza. Modified T cells persisted for over 12 months, with eventual recovery of thymic derived donor T cells after one year and normalisation of immunity. P3 suffered Ligase IV deficiency, a form of radiosensitive SCID. Expansion of modified donor T cells was detected within two weeks of first infusion, but the patient died from mucositis related pulmonary and gastrointestinal haemorrhage before dose escalation.</p

GMP T cell transduction reagents.

<p>GMP T cell transduction reagents.</p

Production of donor HSVTK-CD34 T cells.

<p>Production of donor HSVTK-CD34 T cells.</p

Transduction, enrichment and suicide gene function.

<p>(a) Flow cytometry of peripheral blood lymphocytes after transduction. Cells were activated with anti-CD3/28 beads and underwent two rounds of exposure to vector before removal of activation beads and magnetic bead enrichment using a CliniMacs device. (b) Transduced T cells were enriched (CD34+) to >90% purity for all three products. (c) Upon exposure to the prodrug Ganciclovir (GCV, 10 uM), engineered cells from all three donors had reduced survival compared to non-modified controls (P<0.001). Means of triplicate wells and standard error of means are shown.</p