Background Antithymocyte globulin (ATG) has been increasingly used to prevent graft-vs-host disease (GVHD), however, its impact on immune reconstitution is relatively unknown. Here we studied (1) immune reconstitution after ATG-conditioned hematopoietic cell transplantation (HCT), (2) determined factors influencing the reconstitution, and (3) compared it to non-ATG-conditioned HCT. Methods Immune cell subset counts were determined at 1–24 months posttransplant in 125 HCT recipients who received ATG during conditioning. The subset counts were also determined in 46 non-ATG-conditioned patients (similarly treated). Results (1) Reconstitution after ATG-conditioned HCT was fast for innate immune cells, intermediate for B cells and CD8 T cells, and very slow for CD4 T cells and invariant NKT (iNKT) cells. (2) Faster reconstitution after ATG-conditioned HCT was associated with higher number of cells of the same subset transferred with the graft in case of memory B cells, naïve CD4 T cells, naïve CD8 T cells, iNKT cells and myeloid dendritic cells; lower recipient age in case of naïve CD4 T cells and naïve CD8 T cells; cytomegalovirus recipient seropositivity in case of memory/effector T cells; absence of GVHD in case of naïve B cells; lower ATG serum levels in case of most T cell subsets including iNKT cells, and higher ATG levels in case of NK cells and B cells. (3) Compared to non-ATG-conditioned HCT, reconstitution after ATG-conditioned HCT was slower for CD4 T cells, and faster for NK cells and B cells. Conclusions ATG worsens reconstitution of CD4 T cells but improves reconstitution of NK and B cells.

Graft-versus-host disease (GVHD) is a major transplantation complication. The purpose of this study was to measure immune cell subsets by flow cytometry early after transplantation (before median day of GVHD onset) to identify subsets that may play a role in GVHD pathogenesis. We also measured the subsets later after transplantation to determine which subsets may be influenced by GVHD or its treatment. We studied 219 patients. We found that acute GVHD (aGVHD) was preceded by high counts of CD4 T cells and CD8 T cells. It was followed by low counts of total and naive B cells, total and cytolytic NK cells, and myeloid and plasmacytoid dendritic cells. Chronic GVHD (cGVHD) was preceded by low counts of memory B cells. In conclusion, both CD4 and CD8 T cells appear to play a role in the pathogenesis of aGVHD. Generation of B cells, NK cells, and dendritic cells may be hampered by aGVHD and/or its treatment. Memory B cells may inhibit the development of cGVHD.

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More cytomegalovirus (CMV)-specific T cells are transferred with grafts from CMV seropositive than seronegative donors. We hypothesized that seropositive recipients of grafts from seropositive donors (D+R+) have higher counts of CMV-specific T cells than seropositive recipients of grafts from seronegative donors (D-R+), and that this is clinically relevant in the setting of in vivo T cell depletion using rabbit-antihuman thymocyte globulin (ATG). We reviewed charts of 298 ATG-conditioned, seropositive recipients for CMV reactivation (pp65 antigenemia or CMV DNAemia above institutional threshold for preemptive therapy), recurrent CMV reactivation, CMV disease, and death. In 77 of these patients, we enumerated CMV-specific T cells. Median follow-up was 564 days. CMV-specific CD4+ and, to a lesser degree, CD8+ T cell counts were higher in D+R+ than D-R+ patients. D+R+ patients had lower cumulative incidence of CMV reactivation (21% versus 48%, P < .001), recurrent reactivation (4% versus 15%, P = .003), CMV disease (3% versus 13%, P = .005) and mortality (42% versus 56%, P = .006). We conclude that in the setting of in vivo T cell depletion using ATG, seropositive donors should be used for seropositive recipients. For scenarios where only seronegative donors are available, strategies to improve CMV-specific immunity (e.g., donor vaccination) should be explored.

The largest study on post-allogeneic hematopoietic cell transplant lymphoproliferative disorder (PTLD) epidemiology showed a cumulative incidence of 1.7% in patients receiving antithymocyte globulin (ATG). We had noted an apparently higher incidence in our transplant recipients whose conditioning included ATG. Therefore, we formally determined the incidence of PTLD through chart review. We also evaluated whether counts of EBV-specific T lymphocytes measured by cytokine flow cytometry could identify patients at risk of developing PTLD. Among 307 allogeneic transplant recipients, 25 (8.1%) developed PTLD. This was biopsy proven in 11 patients, and was fatal in seven patients. Patient age, EBV serostatus, donor type/match or GVHD did not influence PTLD risk significantly. Median onset of PTLD was 55 (range, 28-770) days post transplant. Day 28 EBV-specific T lymphocyte counts were not significantly different in 11 patients who developed PTLD and 31 non-PTLD patients matched for published risk factors for PTLD. In summary, when using conditioning with thymoglobulin 4.5 mg/kg, the incidence of PTLD is relatively high and cannot be predicted by day 28 cytokine flow cytometry-determined EBV-specific T lymphocyte counts. Thus, in this scenario PTLD prevention may be warranted, for example, using EBV DNAemia monitoring with preemptive therapy.

Anti-thymocyte globulin (ATG) is polyclonal, containing Ab specificities capable of binding to various immune-cell subsets implicated in the pathogenesis of GVHD, including T cells, B cells, natural killer cells, monocytes/macrophages, neutrophils and DC. We wished to determine which ATG specificities are important for GVHD prevention. We measured day 7 serum levels of 23 ATG specificities in 120 hematopoietic cell transplant recipients whose myeloablative conditioning included 4.5 mg/kg ATG (thymoglobulin). High levels of ATG specificities capable of binding to T-and B-cell subsets were associated with a low likelihood of acute GVHD (aGVHD). High levels of these ATG specificities were associated with increased rates of viral but not bacterial or fungal infections. They were not associated with an increased risk of malignancy relapse; on the contrary, high levels of ATG specificities capable of binding to regulatory T cells and invariant NKT cells were associated with a low risk of relapse. In conclusion, high levels of ATG antibodies to Ag(s) expressed on T and B cells are associated with a low risk of aGVHD and a high risk of viral but not bacterial or fungal infections. These antibodies have neutral or beneficial effects on relapse.

1981 Introduction: Immune reconstitution after HCT is important for curbing infections and malignancy. ATG has been increasingly used to prevent graft-vs-host disease (GVHD), however, its impact on immune reconstitution has not been well studied. Here we studied (1) immune reconstitution after ATG-conditioned HCT, (2) compared it to non-ATG-conditioned HCT, and (3) determined factors influencing the immune reconstitution. Patients and Methods: Immune subset cell counts were determined on day 28, 56, 84, 180, 365 and 730 post transplant in 125 recipients of allogeneic filgrastim-mobilized blood stem cells who received ATG (Thymoglobulin, 4.5 mg/kg) during conditioning. The subset counts were also determined in 47 non-ATG-conditioned patients (otherwise similarly treated). Subset counts (in blood) and ATG levels (in serum) were quantified by flow cytometry. Mann-Whitney rank sum test was used to compare subset counts (1) in ATG-conditioned patients vs donors, (2) in ATG-conditioned patients vs non-ATG-conditioned patients, and (3) between subgroups of ATG-conditioned patients; Spearman rank correlation test was used to determine associations between subset counts and ordinal variables like ATG levels. Results: (1) After ATG-conditioned HCT, the counts of the following subsets normalized (became not significantly lower than in donors) by day 28: NK cells, monocytes, myeloid dendritic cells (MDCs), and plasmacytoid dendritic cells (PDCs). The counts of the following subsets normalized by day 84: memory/effector CD8 T cells, and CD4−CD8− T cells. The counts of naïve B cells normalized by day 180. The counts of the following subsets have not normalized by day 365 or 730: memory B cells (both isotype switched and unswitched), both naïve and memory/effector CD4 T cells, naïve CD8 T cells, CD4+CD8+ T cells, and invariant NKT (iNKT) cells. (2) Compared to non-ATG-conditioned HCT, counts of B cells, CD4 T cells and CD8 T cells were significantly lower after ATG-conditioned HCT on day 28. Thereafter, recovery of both naïve and memory B cells and memory/effector CD8 T cells was significantly faster in ATG-conditioned patients, leading to higher total B and higher total CD8 T cell counts on day 84 (Figure). On the contrary, recovery of naïve CD8 T cells and both naïve and memory/effector CD4 T cells was significantly slower, the latter leading to low total CD4 T cell counts throughout the first year (Figure). (3) Reconstitution after ATG-conditioned HCT was influenced by (a) the number of cells of the same subset transferred with the graft in case of increased memory B cells, naïve CD4 T cells, naïve CD8 T cells, iNKT cells and MDCs, (b) age of recipient in case of decreased naïve CD4 T cells and naïve CD8 T cells, (c) cytomegalovirus (CMV) serostatus of recipient in case of increased memory/effector T cells, (d) GVHD in case of increased naïve B cells, and (e) day 7 or 28 ATG levels in case of decreased T cell subsets. Conclusion: (1) Reconstitution after ATG conditioned HCT is very fast for NK cells, monocytes, MDCs and PDCs, fast for memory/effector CD8 T cells and CD4−CD8− T cells, slow for naïve B cells, and very slow for memory B cells, both naïve and memory/effector CD4 T cells, naïve CD8 T cells, CD4+CD8+ T cells and iNKT cells. (2) Compared to no ATG, the patients conditioned with ATG have lower counts of B and T cells on day 28. Thereafter, the ATG-conditioned patients have faster recovery of both naïve and memory B cells and memory/effector CD8 T cells, and slower recovery of both naïve and memory/effector CD4 T cells and naïve CD8 T cells. (3) Similar to what has been described for non-ATG-conditioned HCT, reconstitution after ATG-conditioned HCT is influenced by the number of the immune cells transferred with the graft, recipient age, recipient CMV serostatus and GVHD. Moreover, the reconstitution after ATG-conditioned HCT is influenced by ATG clearance. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic cell transplant (HCT) recipients are immunocompromised and thus predisposed to infections. We set out to determine the deficiency of which immune cell subset(s) may predispose to postengraftment infections. We determined day 28, 56, 84, and 180 blood counts of multiple immune cell subsets in 219 allogeneic transplant recipients conditioned with busulfan, fludarabine, and Thymoglobulin. Deficiency of a subset was considered to be associated with infections if the low subset count was significantly associated with subsequent high infection rate per multivariate analysis in both discovery and validation cohorts. Low counts of monocytes (total and inflammatory) and basophils, and low IgA levels were associated with viral infections. Low plasmacytoid dendritic cell (PDC) counts were associated with bacterial infections. Low inflammatory monocyte counts were associated with fungal infections. Low counts of total and naive B cells, total and CD56(high) natural killer (NK) cells, total and inflammatory monocytes, myeloid dendritic cells (MDCs), PDCs, basophils and eosinophils, and low levels of IgA were associated with any infections (due to any pathogen or presumed). In conclusion, deficiencies of B cells, NK cells, monocytes, MDCs, PDCs, basophils, eosinophils, and/or IgA plasma cells appear to predispose to postengraftment infections.