Depletion of CD45RA+ T cells: Advantages and disadvantages of different purification methods

Bremm, Melanie; Krastel, Theresa; Cappel, Claudia; Zimmermann, Olga; Pfeffermann, Lisa-Marie; Katzki, Verena; Bönig, Halvard; Schäfer, Richard; Rettinger, Eva; Merker, Michael; Bremm, Sebastian; Schaefer, Kirsten; Klingebiel, Thomas; Soerensen, Jan; Bader, Peter; Huenecke, Sabine

doi:10.1016/j.jim.2021.112960

Cited by 3 publications

(11 citation statements)

References 43 publications

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“…Results and figure are part of a publication by Bremm et al to which I contributed as a co-author. 65…”

Section: Figure 12 Cd45ra Co-expression Measurements Cd-45ra Co-expr...mentioning

confidence: 99%

“…CD45RA-Pacific Blue (Graph 1) showed inadequate separation between CD45RA+ and CD45RA-cells after additional contact with the depletion antibody, while CD45RA-FITC (plot 2) showed better separation and CD45RA-PE (plot 3) shows the most adequate separation. Results and figure are part of a publication by Bremm et al to which I contributed as a co-author 65.…”

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confidence: 99%

“…To ensure valid measurements of remaining CD45RA+ T cells after depletion, a spike control (plot 3) was established. For this purpose, a 1:6 mixture of cells from CCB and NTCB was measured after CD45RA depletion Results and figure are part of a publication by Bremm et al to which I contributed as a co-author 65.…”

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confidence: 99%

“…For the two-step approach only TM recovery data are shown because the starting product did not contain SC. Results and figure are part of a publication by Bremm et al to which I contributed as a coauthor 65.…”

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confidence: 99%

“…2.Shows the frequency distribution of detected virus-specific T cells after CD45RA depletion. Measurementswere performed in the one-step (n=3) and two-step (n=4) approaches (EBV: n=15, CMV: n=5, ADV: n=5).Results are part of a publication byBremm et al to which I contributed as a co-author 65.…”

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Depletion of CD45RA+ naïve T cells for haploidentical stem cell transplantations: graft generation under good manufacturing practice and establishment of a flow cytometric quality control for clinical-scale grafts

Krastel¹

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In haploidentical stem cell transplantation (SCT), achieving a balance between graft versus host disease (GvHD), graft versus leukemia effect (GvL) and bridging the vulnerable phase of aplasia against viral infections is still a challenge. Graft preparation strategies attempt to achieve this balance by removing and retaining harmful and helpful cells. At this point it is known that T cell subpopulations hold different properties concerning GvHD promotion and immunocompetence towards pathogens. CD45RA+ naïve T cells show the greatest, while CD45RO+ memory T cells show less alloreactive potential but provide immunocompetence. CD45RA depletion is a promising new approach to graft processing that potentially combines GvHD prevention, GvL promotion and transfer of immunological competence by removing potentially harmful CD45RA+ naïve T cells and retaining CD45RO+ memory cells. This work focused on manufacturing CD45RA-depleted grafts within a one- or two-step approach, as well as a feasibility assessment of the process and the establishment of a 10-color fluorescence activated cell sorting (FACS) measurement panel for clinical-scale graft generation. CD45RA depletions were conducted from granulocyte-colony stimulated factor (G-CSF) mobilized peripheral blood stem cells (PBSC) applying two different strategies, direct depletion of CD45RA+ cells (one-step approach), or depletion following preceding CD34 selection. A 10-color FACS measurement panel was established ensuring quality control and enabling preliminary data acquisition on CD45RA co-expression for cell loss estimations. Residual virus-specific T cells after depletion were measured using MHC multimers. It was observed that the depletion antibody occupied the cell binding sites, resulting in insufficient binding of the fluorescent dye for subsequent FACS measurement. Therefore, three FACS antibodies were tested and compared, and CD45RA-PE (clone:2H4) was found to be the best choice for reliable cell detection. To further characterize residual T cells, two homing markers, CD62L and CCR7, were compared, with particular attention paid to the expression of the surface markers after cooling. Both markers were complementary to each other, resulting in the decision to include an additional FACS measuring tube whenever samples are cooled or further T cell characterization is needed. With a median log depletion of -3.9 (one-step) and -3.8 (two-step) data showed equally efficient removal of CD45RA+CD3+ T cells for both approaches. Close to complete B cell removal was obtained without additional reagent use. However, also close to complete NK cell loss occurred due to high CD45RA co-expression. Stem cells recovered at a median of 52% (range: 49.7 - 67.2%) after one-step CD45RA depletion. CD45RO+ memory T cells recovery was statistically not differing between both approaches. Virus-specific T cells were detectable after depletion, suggesting that virus-specific immunocompetence is transferable. In conclusion, CD45RA depletions are equally feasible for both approaches when performed from fresh, non-cryopreserved starting products, show reliable reduction of CD45RA and B cells, but also result in co-depletion of NK cells. Stem cell recovery and NK cell losses must be considered carefully especially regarding overcoming HLA barriers, pathogen protection during aplasia, early engraftment an GvL. Therefore, a combination of CD45RA-depleted products with already established other processing methods to ensure sufficient stem and NK cells is desirable to allow high clinical flexibility.

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“…Results and figure are part of a publication by Bremm et al to which I contributed as a co-author. 65…”

Section: Figure 12 Cd45ra Co-expression Measurements Cd-45ra Co-expr...mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

mentioning

confidence: 99%

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Depletion of CD45RA+ naïve T cells for haploidentical stem cell transplantations: graft generation under good manufacturing practice and establishment of a flow cytometric quality control for clinical-scale grafts

Krastel¹

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Site-specific differences in T lymphocyte composition of the gastric mucosa after Helicobacter pylori eradication

et al. 2022

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In our earlier work, we revealed that inflammation of the lesser curvature of the gastric body and antrum could constitute independent risk factors for gastric cancer development, while inflammation of the greater curvature was not. The aims of this study were as follows: first, to reveal the differences between T lymphocyte populations of the gastric antrum and the greater and lesser curvatures of the gastric body in patients after Helicobacter pylori eradication; second, to analyze the correlation between the composition of the stomach-resident T lymphocytes and time from H. pylori eradication; and third, to evaluate the sex differences in T lymphocyte subsets after H. pylori eradication. To investigate site-specific differences in stomach-resident T lymphocytes after H. pylori eradication, we performed flow cytometry analysis on samples taken from the gastric antrum, greater curvature of the gastric body, and lesser curvature of the gastric body of 20 patients. We also analyzed the correlation between the composition of the stomach-resident T lymphocytes and the time from H. pylori eradication. The lymphocyte subsets of the antrum and lesser curvature of the body were similar. In contrast, compared to those in the greater curvature of the gastric body, CD4+/CD3+ lymphocyte subsets (43.8 ± 19.4% vs 31.7 ± 14.6%) were elevated in the lesser curvature of the body, whereas CD8+/CD3+ (67.1 ± 21.3% vs 80.4 ± 12.0%), CD7+/CD3+ (91.2 ± 4.6% vs 93.7 ± 3.8%), CCR4+/CD3+ (7.7 ± 8.1% vs 10.4 ± 7.0%), CD45RA+/CD3+CD4+ (27.2 ± 24.8% vs 39.5 ± 20.8%), and CD45RA+/CD3+CD4− (14.2 ± 11.1% vs 18.7 ± 11.5) were lower. Linear regression analysis showed a negative correlation between the time after H. pylori eradication and CD4+/CD3+ (P < .05, R2 = 0.198). There were no significant differences between men and women with respect to the lymphocyte populations. These results indicate that there are site-specific differences in lymphocyte composition in the stomach after H. pylori eradication.

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Familial CD45RA– T cells to treat severe refractory infections in immunocompromised patients

Sanz

Parente²,

Ferreras³

et al. 2023

Front. Med.

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BackgroundImmunocompromised patients are susceptible to high-risk opportunistic infections and malignant diseases. Most antiviral and antifungal drugs are quite toxic, relatively ineffective, and induce resistance in the long term. The transfer of pathogen-specific Cytotoxic T-Lymphocytes has shown a minimal toxicity profile and effectiveness in treating Cytomegalovirus, Adenovirus, Epstein - Barr virus, BK Virus and Aspergillus infections, but this therapy have the main limitations of regulatory issues, high cost, and absence of public cell banks. However, CD45RA– cells containing pathogen-specific memory T-cells involve a less complex manufacturing and regulatory process and are cheaper, feasible, safe, and potentially effective.MethodsWe present preliminary data from six immunocompromised patients: four who had severe infectious diseases and two who had EBV lymphoproliferative disease. All of them underwent multiple safe familial CD45RA– T-cell infusions as adoptive passive cell therapy, containing Cytomegalovirus, Epstein - Barr virus, BK virus, and Aspergillus-specific memory T-cells. We also present the method for selecting the best donors for CD45RA– cells in each case and the procedure to isolate and store these cells.ResultsThe infusions were safe, there was no case of graft-versus host disease, and they showed a clear clinical benefit. The patients treated for BK virus nephritis, Cytomegalovirus encephalitis, Cytomegalovirus reactivation, and disseminated invasive aspergillosis experienced pathogen clearance, complete resolution of symptoms in 4-6 weeks and a lymphocyte increase in 3 of 4 cases after 3–4 months. Donor T cell transient microchimerism was detected in one patient. The two patients treated for EBV lymphoproliferative disease underwent chemotherapy and several infusions of CD45RA– memory T-cells containing EBV cytotoxic lymphocytes. Donor T-cell microchimerism was observed in both patients. The viremia cleared in one of the patients, and in the other, despite the viremia not clearing, hepatic lymphoproliferative disease remained stable and was ultimately cured with EBV-specific Cytotoxic T-Lymphocytes.ConclusionThe use of familial CD45RA– T-cells containing specific Cytotoxic T-lymphocytes is a feasible, safe and potential effective approach for treating severe pathogen infections in immunocompromised patients through a third party donor. Furthermore, this approach might be of universal use with fewer institutional and regulatory barriers.

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Depletion of CD45RA+ T cells: Advantages and disadvantages of different purification methods

Cited by 3 publications

References 43 publications

Depletion of CD45RA+ naïve T cells for haploidentical stem cell transplantations: graft generation under good manufacturing practice and establishment of a flow cytometric quality control for clinical-scale grafts

Depletion of CD45RA+ naïve T cells for haploidentical stem cell transplantations: graft generation under good manufacturing practice and establishment of a flow cytometric quality control for clinical-scale grafts

Site-specific differences in T lymphocyte composition of the gastric mucosa after Helicobacter pylori eradication

Familial CD45RA– T cells to treat severe refractory infections in immunocompromised patients

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