Annals of Stem Cell Research (ASCR)
ISSN: 2689-8659 | Vol. 6, Issue 1, 2023, pp. 25-30

Graft-versus-Host Disease Prophylaxis and Immune Reconstitution After Allogeneic Transplantation

Georgiou E1, Laine N2, Kaczmarek T3

1Department of Clinical Immunology, Aristotle University of Thessaloniki, Thessaloniki, Greece
2Department of Haematology and Stem Cell Transplantation, University of Helsinki, Helsinki, Finland
3Department of Molecular Cell Biology, Medical University of Warsaw, Warsaw, Poland
DOI: 10.5281/zenodo.22238007 | Published: 2023-06-07 | Article page and PDF

Abstract

Background: Prophylaxis against graft-versus-host disease has changed more in the past decade than in the preceding three,

and each regimen shapes immune recovery differently.

Objective: To review the randomised evidence for current prophylaxis regimens and what each does to immune

reconstitution.

Methods: Narrative review of randomised trials, registry analyses and reconstitution studies in allogeneic transplantation.

Findings: Post-transplant cyclophosphamide raised one-year graft-versus-host-disease-free relapse-free survival from 34.9%

to 52.7% (hazard ratio 0.64), while overall survival, relapse and transplantation-related death did not differ substantially.

Anti-lymphocyte globulin halved chronic disease with identical survival, and a registry analysis of 5,209 matched-sibling

transplants found the two regimens indistinguishable except in relapse.

Conclusions: The gains are in composite endpoints. Whether they represent better transplantation or better-scored

transplantation is the open question.

Keywords: graft-versus-host disease; cyclophosphamide; immune reconstitution; allogeneic transplantation; prophylaxis

Introduction

Allogeneic transplantation cures by an immune mechanism and injures by the same one. The donor graft attacks residual malignancy and, indiscriminately, the recipient’s skin, gut and liver, and every prophylaxis regimen attempts to separate the two [1].

For three decades the standard was a calcineurin inhibitor with methotrexate, established when the combination proved superior to cyclosporine alone [2]. What displaced it originated in haploidentical transplantation, where high-dose cyclophosphamide after graft infusion made transplantation across a full haplotype barrier feasible [3], then worked as single-agent prophylaxis [4], producing outcomes equivalent to contemporaneous matched transplantation [5]; the approaches have been reviewed [6]. The mechanism is not simply deletion of alloreactive cells [7].

This review considers what the randomised evidence supports, and what each regimen costs in immune recovery.

Materials and Methods

Records were retrieved from Crossref, OpenAlex, Europe PMC and publisher pages for the terms graft-versus-host disease, post-transplant cyclophosphamide, anti-thymocyte globulin, ruxolitinib and immune reconstitution. Priority was given to randomised trials and to registry analyses large enough to be informative. Every bibliographic record was confirmed at source, and quantitative values were taken from the source rather than from secondary citation.

Post-transplant cyclophosphamide in matched donors

The trial that changed practice compared cyclophosphamide, tacrolimus and mycophenolate against tacrolimus and methotrexate in reduced-intensity matched transplantation. At one year, adjusted graft-versus-host-disease-free relapse-free survival was 52.7% (95% CI 45.8–59.2) with the experimental regimen and 34.9% (95% CI 28.6–41.3) with the standard, hazard ratio 0.64 (95% CI 0.49–0.83, p = 0.001), among 214 and 217 patients respectively [8]. Patient-reported outcomes and two-year results were published subsequently [9].

Read the same abstract to the end and the qualification appears: overall and disease-free survival, relapse, transplantation-related death and engraftment did not differ substantially between the groups. The improvement is in a composite that counts graft-versus-host disease as an event.

Supporting randomised evidence is consistent in shape. A phase II trial compared three regimens against tacrolimus and methotrexate [10], a Dutch trial found benefit for the cyclophosphamide arm [11], and an Australian myeloablative trial reported three-year survival free of graft-versus-host disease and relapse of 49.3% against 14.1%, hazard ratio 0.419, in 134 adults — with improved relapse-free but not overall survival [12]. A registry analysis of 5,209 matched related transplants found the approaches indistinguishable except in relapse, 26.2%

Figure 1: Composite endpoint against overall survival in three randomised comparisons. Every regimen improves the composite; none improves survival, and CD34 selection worsens it. Data from Bolanos-Meade et al. [8], Kroeger et al. [16] and Luznik et al. [23].
Figure 1: Composite endpoint against overall survival in three randomised comparisons. Every regimen improves the composite; none improves survival, and CD34 selection worsens it. Data from Bolanos-Meade et al. [8], Kroeger et al. [16] and Luznik et al. [23].

Anti-thymocyte and anti-lymphocyte globulin have been tested in randomised trials with a consistent result. Adding anti-T-cell globulin reduced chronic disease [14], as did pretreatment with anti-thymocyte globulin [15]. The clearest arithmetic comes from the anti-lymphocyte globulin trial: chronic disease at two years fell from 68.7% to 32.2%, p < 0.001, with the composite endpoint rising from 16.8% to 36.6%, p = 0.005 — and two-year overall survival of 74.1% against 77.9%, p = 0.46 [16]. A double-blind phase III trial assessed the same question [17].

Serotherapy has a dose-exposure relationship with immune recovery that is measurable and clinically relevant: anti-thymocyte globulin exposure is associated with CD4 reconstitution in paediatric transplantation [18], which is the mechanism by which it trades chronic disease against infection.

Costimulation blockade and treatment of established disease

Abatacept reduced severe acute disease, with day-180 severe-disease-free survival of 93.2% against 82% (p = 0.05) randomised, and 97.7% against 58.7% in a mismatched cohort against registry controls [19].

For established steroid-refractory disease, ruxolitinib is now standard. A phase II study established activity [20]; the acute trial gave a day-28 response of 62% against 39% [21]; the chronic trial gave a week-24 response of 49.7% against 25.6% (p < 0.001), with median failure-free survival exceeding 18.6 months against 5.7 [22].

T-cell depletion was tested against both alternatives. CD34 selection reduced moderate and severe chronic disease markedly, hazard ratio 0.25 (95% CI 0.12–0.52), yet two-year overall survival was 60.1% against 76.1%, hazard ratio 1.74 (p = 0.02), driven by transplant-related mortality [23]. Here less chronic disease translated into worse survival.

Immune reconstitution

Recovery follows a stereotyped order — natural killer cells within 30 to 100 days, T cells by about 100 days, B cells over one to two years, with CD4 counts around 200/μl at three months [24]. Thymic output sets the pace and can be measured [25]; regeneration after immunological injury has been reviewed [26]. Graft source matters [27], and delayed reconstitution is associated with higher mortality [28].

Prophylaxis regimens shape this differently. Post-transplant cyclophosphamide impairs early T- and natural killer cell reconstitution [29]. A prospective comparison against anti-thymocyte globulin found lower day-30 counts with cyclophosphamide — total lymphocytes 473 against 924/μl (p = 0.0004), T cells 82 against 238/μl (p = 0.022), natural killer cells 96 against 385/μl (p < 0.001) — while by day 180 B-cell recovery favoured it [30].

Discussion

Three conclusions follow. First, post-transplant cyclophosphamide improves a composite endpoint in matched-donor transplantation on good randomised evidence, and has not been shown to improve survival. Second, the same is true of serotherapy and, more starkly, of CD34 selection, where less chronic disease came with worse survival. Third, the regimens differ substantially in early immune recovery, which is the plausible route by which a reduction in graft-versus-host disease fails to become a reduction in mortality.

Table 1: Randomised prophylaxis trials: the composite endpoint against overall survival.
Table 1: Randomised prophylaxis trials: the composite endpoint against overall survival.

Trial Comparison Composite endpoint Overall survival

BMT CTN CD34 selection cGVHD HR 0.25 60.1% vs 1301 [23] vs Tac/MTX (0.12-0.52) 76.1%; HR 1.74 EBMT PTCy vs rATG, GRFS 46.5% vs 42.7%; 68.5% vs registry [13] n = 5,209 p = 0.32 64.8%; p = 0.92 Whether the composite endpoint is a legitimate surrogate or a construct that rewards one arm of the trade-off while concealing its infectious and relapse cost is the field’s genuine disagreement, and it is not resolved by any trial published so far.

This is a narrative review without systematic search or risk-of-bias assessment. Several quantitative results could not be confirmed at source and are therefore not reported, and trials differ in conditioning intensity, donor type and graft source in ways that prevent pooling.

Conclusion

Post-transplant cyclophosphamide has moved from a haploidentical technique to a matched-donor standard on the strength of randomised evidence for a composite endpoint, without a demonstrated survival benefit. Serotherapy shows the same pattern and CD34 selection the inverse one. The regimens are not interchangeable from the recipient’s point of view: they produce measurably different early lymphocyte recovery, and reconstitution kinetics are themselves associated with mortality. Trials should report immune reconstitution alongside disease incidence, and should present the components of any composite endpoint separately, so that a reduction in graft-versus-host disease can be weighed against what it costs.

Figure 2: Lymphocyte subsets at day 30 after unrelated-donor transplantation with post-transplant cyclophosphamide against anti-thymocyte globulin. The delay is substantial and is the plausible route by which less graft-versus-host disease fails to become better
Figure 2: Lymphocyte subsets at day 30 after unrelated-donor transplantation with post-transplant cyclophosphamide against anti-thymocyte globulin. The delay is substantial and is the plausible route by which less graft-versus-host disease fails to become better

survival. Data from Kerbauy et al. [30].

Acknowledgements

The authors thank the library staff who assisted with retrieval of the primary literature reviewed here.

Funding

This review received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.

Conflict of Interest

The authors declare that they have no competing interests relevant to the content of this review.

Ethics Statement

This article reviews previously published studies and contains no new studies with human participants or animals performed by any of the authors. Ethical approval was therefore not required.

References

  1. Zeiser, R., Blazar, B.R. (2017) Acute graft-versus-host disease - biologic process, prevention, and therapy. N Engl J Med, 377(22): 2167-2179.
  2. Storb, R., Deeg, H.J., Whitehead, J., Appelbaum, F., Beatty, P., Bensinger, W., et al. (1986) Methotrexate and cyclosporine compared with cyclosporine alone for prophylaxis of acute graft versus host disease after marrow transplantation for leukemia. N Engl J Med, 314(12): 729-735.
  3. Luznik, L., O’Donnell, P.V., Symons, H.J., Chen, A.R., Leffell, M.S., Zahurak, M., et al. (2008) HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide. Biol Blood Marrow Transplant, 14(6): 641-650.
  4. Luznik, L., Bolanos-Meade, J., Zahurak, M., Chen, A.R., Smith, B.D., Brodsky, R., et al. (2010) High-dose cyclophosphamide as single-agent, short-course prophylaxis of graft-versus-host disease. Blood, 115(16): 3224-3230.
  5. Bashey, A., Zhang, X., Sizemore, C.A., Manion, K., Brown, S., Holland, H.K., et al. (2013) T-cell-replete HLA-haploidentical hematopoietic transplantation for hematologic malignancies using post-transplantation cyclophosphamide results in outcomes equivalent to those of contemporaneous HLA-matched related and unrelated donor transplantation. J Clin Oncol, 31(10): 1310-1316.
  6. Kanakry, C.G., Fuchs, E.J., Luznik, L. (2016) Modern approaches to HLA-haploidentical blood or marrow transplantation. Nat Rev Clin Oncol, 13(1): 10-24.
  7. Nunes, N.S., Kanakry, C.G. (2019) Mechanisms of graft-versus-host disease prevention by post-transplantation cyclophosphamide: an evolving understanding. Front Immunol, 10: 2668.
  8. Bolanos-Meade, J., Hamadani, M., Wu, J., Al Malki, M.M., Martens, M.J., Runaas, L., et al. (2023) Post-transplantation cyclophosphamide-based graft-versus-host disease prophylaxis. N Engl J Med, 388(25): 2338-2348.
  9. Holtan, S.G., Bolanos-Meade, J., Al Malki, M.M., Wu, J., Kitko, C.L., Reshef, R., et al. (2025) Improved patient-reported outcomes with post-transplant cyclophosphamide: a quality-of-life evaluation and 2-year outcomes of BMT CTN 1703. J Clin Oncol, 43(8): 912-918.
  10. Bolanos-Meade, J., Reshef, R., Fraser, R., Fei, M., Abhyankar, S., Al-Kadhimi, Z., et al. (2019) Three prophylaxis regimens versus tacrolimus and methotrexate for prevention of graft-versus-host disease with haemopoietic cell transplantation with reduced-intensity conditioning: a randomised phase 2 trial with a non-randomised contemporaneous control group (BMT CTN 1203). Lancet Haematol, 6(3): e132-e143.
  11. Broers, A.E.C., de Jong, C.N., Bakunina, K., Hazenberg, M.D., van Marwijk Kooy, M., de Groot, M.R., et al. (2022) Posttransplant cyclophosphamide for prevention of graft-versus-host disease: results of the prospective randomized HOVON-96 trial. Blood Adv, 6(11): 3378-3385.
  12. Curtis, D.J., Patil, S.S., Reynolds, J., Purtill, D., Lewis, C., Ritchie, D.S., et al. (2025) Graft-versus-host disease prophylaxis with cyclophosphamide and cyclosporin. N Engl J Med, 393(3): 243-254.
  13. Penack, O., Abouqateb, M., Peczynski, C., Boreland, W., Benakli, M., Kroger, N., et al. (2025) GVHD prophylaxis in matched related stem cell transplantation: why post-transplant cyclophosphamide can be recommended. A study by the EBMT transplant complications working party. Leukemia, 39(6): 1503-1511.
  14. Finke, J., Bethge, W.A., Schmoor, C., Ottinger, H.D., Stelljes, M., Zander, A.R., et al. (2009) Standard graft-versus-host disease prophylaxis with or without anti-T-cell globulin in haematopoietic cell transplantation from matched unrelated donors: a randomised, open-label, multicentre phase 3 trial. Lancet Oncol, 10(9): 855-864.
  15. Walker, I., Panzarella, T., Couban, S., Couture, F., Devins, G., Elemary, M., et al. (2016) Pretreatment with anti-thymocyte globulin versus no anti-thymocyte globulin in patients with haematological malignancies undergoing haemopoietic cell transplantation from unrelated donors: a randomised, controlled, open-label, phase 3, multicentre trial. Lancet Oncol, 17(2): 164-173.
  16. Kroger, N., Solano, C., Wolschke, C., Bandini, G., Patriarca, F., Pini, M., et al. (2016) Antilymphocyte globulin for prevention of chronic graft-versus-host disease. N Engl J Med, 374(1): 43-53.
  17. Soiffer, R.J., Kim, H.T., McGuirk, J., Horwitz, M.E., Johnston, L., Patnaik, M.M., et al. (2017) Prospective, randomized, double-blind, phase III clinical trial of anti-T-lymphocyte globulin to assess impact on chronic graft-versus-host disease-free survival in patients undergoing HLA-matched unrelated myeloablative hematopoietic cell transplantation. J Clin Oncol, 35(36): 4003-4011.
  18. Admiraal, R., van Kesteren, C., Jol-van der Zijde, C.M., Lankester, A.C., Bierings, M.B., Egberts, T.C.G., et al. (2015) Association between anti-thymocyte globulin exposure and CD4+ immune reconstitution in paediatric haemopoietic cell transplantation: a multicentre, retrospective pharmacodynamic cohort analysis. Lancet Haematol, 2(5): e194-e203.
  19. Watkins, B., Qayed, M., McCracken, C., Bratrude, B., Betz, K., Suessmuth, Y., et al. (2021) Phase II trial of costimulation blockade with abatacept for prevention of acute GVHD. J Clin Oncol, 39(17): 1865-1877.
  20. Jagasia, M., Perales, M.A., Schroeder, M.A., Ali, H., Shah, N.N., Chen, Y.B., et al. (2020) Ruxolitinib for the treatment of steroid-refractory acute GVHD (REACH1): a multicenter, open-label phase 2 trial. Blood, 135(20): 1739-1749.
  21. Zeiser, R., von Bubnoff, N., Butler, J., Mohty, M., Niederwieser, D., Or, R., et al. (2020) Ruxolitinib for glucocorticoid-refractory acute graft-versus-host disease. N Engl J Med, 382(19): 1800-1810.
  22. Zeiser, R., Polverelli, N., Ram, R., Hashmi, S.K., Chakraverty, R., Middeke, J.M., et al. (2021) Ruxolitinib for glucocorticoid-refractory chronic graft-versus-host disease. N Engl J Med, 385(3): 228-238.
  23. Luznik, L., Pasquini, M.C., Logan, B., Soiffer, R.J., Wu, J., Devine, S.M., et al. (2022) Randomized phase III BMT CTN trial of calcineurin inhibitor-free chronic graft-versus-host disease interventions in myeloablative hematopoietic cell transplantation for hematologic malignancies. J Clin Oncol, 40(4): 356-368.
  24. Ogonek, J., Kralj Juric, M., Ghimire, S., Varanasi, P.R., Holler, E., Greinix, H., et al. (2016) Immune reconstitution after allogeneic hematopoietic stem cell transplantation. Front Immunol, 7: 507.
  25. Douek, D.C., Vescio, R.A., Betts, M.R., Brenchley, J.M., Hill, B.J., Zhang, L., et al. (2000) Assessment of thymic output in adults after haematopoietic stem cell transplantation and prediction of T-cell reconstitution. Lancet, 355(9218): 1875-1881.
  26. Velardi, E., Tsai, J.J., van den Brink, M.R.M. (2021) T cell regeneration after immunological injury. Nat Rev Immunol, 21(5): 277-291.
  27. Storek, J., Dawson, M.A., Storer, B., Stevens-Ayers, T., Maloney, D.G., Marr, K.A., et al. (2001) Immune reconstitution after allogeneic marrow transplantation compared with blood stem cell transplantation. Blood, 97(11): 3380-3389.
  28. Bejanyan, N., Brunstein, C.G., Cao, Q., Lazaryan, A., Luo, X., Curtsinger, J., et al. (2018) Delayed immune reconstitution after allogeneic transplantation increases the risks of mortality and chronic GVHD. Blood Adv, 2(8): 909-922.
  29. Rambaldi, B., Kim, H.T., Reynolds, C., Chamling Rai, S., Arihara, Y., Kubo, T., et al. (2021) Impaired T- and NK-cell reconstitution after haploidentical HCT with posttransplant cyclophosphamide. Blood Adv, 5(2): 352-364.
  30. Kerbauy, M.N., Rocha, F.A., Arcuri, L.J., Cunegundes, P.S., Kerbauy, L.N., Machado, C.M., et al. (2025) Immune reconstitution dynamics after unrelated allogeneic transplantation with post-transplant cyclophosphamide compared to classical immunosuppression with anti-thymocyte globulin: a prospective cohort study. Haematologica, 110(3): 640-650.