To the editor:
The absence of cellular immunity is central to the pathogenesis of herpesvirus-mediated diseases after allogeneic hemopoietic stem cell transplantation (HSCT).1,2 For both bone marrow (BM)– and granulocyte-colony stimulating factor–mobilized peripheral blood stem cells (PBSCs) HSCT, donor-derived Epstein-Barr virus (EBV) and cytomegalovirus (CMV) peptide–specific CD8+ T cells clones undergo early expansion and persist long-term, with additional diversification arising from novel antigen-specific clones from donor-derived progenitors.3 Whether BM or PBSC is the superior source of antiviral CD8+ T cells is unclear. Given that PBSC has largely replaced BM as a source of stem cells for HSCT, it is unlikely that herpesvirus effector T-cell reconstitution will ever be compared prospectively. PBSC grafts contain 10 to 30 times more T cells than BM4 and a randomized study found proven viral infections were more frequent in BM than PBSC recipients,5 suggesting viral-specific T-cell immunity is enhanced in PBSC. Recently Moss showed in lung cancer patients that herpesvirus-specific BM-derived CD8+ T cells have unique homing properties relative to herpesvirus-specific CD8+ T cells present in unmobilized peripheral blood (PB).6 Immunodominant EBV-lytic peptide–specific CD8+ T cells were enriched in BM but were reduced for CMV peptide–specific CD8+ T cells relative to PB. EBV-latent peptide–specific CD8+ T cells were equivalent, which has relevance in the context of posttransplantation lymphoproliferative disorder for which impaired EBV-latent CD8+ T-cell immunity is a risk-factor.7 A comparison of herpesvirus-specific cellular immunity in PBSC versus PB has yet to be performed.
We assayed the PBSC of 16 patients and the PB of 26 age-matched healthy volunteers. Although PBSC was obtained in patients and compared with PB of healthy subjects, our previous data indicate that CMV/EBV effector T-cell immunity is not impaired in these patients.8 The study had ethics approval and was conducted in accordance with the Declaration of Helsinki. In line with Moss in BM versus PB,6 CD8+ T cells were significantly increased and naive CD8+ T cells were reduced in PBSC relative to PB. Otherwise, CD8+ T-cell subsets in PBSC relative to PB were strikingly different compared with BM (Table 1). Undifferentiated CD8+ T cells (coexpressing the costimulatory molecules CD27 and CD28) were reduced. In addition, CD8+ T cells expressing the lymphoid tissue homing molecule CD62L were lower which is consistent with recent mobilization of T cells from BM into the circulation. In keeping with this, we observed elevation of the chemokine receptors CXCR3 and CCR5 (markers of migratory effector T cells), but not CXCR6 (which facilitates egress into the lung, liver, and joints9 ), whereas in BM there is enrichment for CXCR3−CCR5+CXCR6+CD8+ T cells. As expected, the normal hierarchy of EBV lytic greater than latent T-cell responses seen in PB (P = .01) was preserved in PBSC (P = .03). Critically and in contrast to BM,6 in PBSC EBV-latent peptide–specific interferon-γ (IFN-γ) secreting CD3+CD8+ T cells were 20-fold lower than in PB (perhaps reflecting the lower EBV viral load in PBSC), whereas EBV-lytic and CMV peptide–specific IFN-γ CD8+ T cells were equivalent to PB. These results imply that within PBSC there is selective recruitment of CD8+ T-cell populations with a distinct functional and homing phenotype. In contrast to BM, EBV-latent but not CMV peptide–specific CD8+ T-cell immunity is impaired in PBSC relative to PB. The data have implications for HSCT and adoptive immunotherapy.
Characteristic . | PBSC . | PB . | P . | Findings of Moss in BM vs PB . | ||
---|---|---|---|---|---|---|
Median (range) . | SE . | Median (range) . | SE . | |||
Age, y | 66 (22-70) | 3.713 | 53 (24-72) | 2.79 | NS | |
Sex, M/F | 11/5 | NA | 13/13 | NA | NS | |
Diagnosis | 7 myeloma, 8 lymphoma, 1 Ewing sarcoma | NA | Healthy | NA | NA | 80% primary lung cancer, 20% secondary lung cancer |
% CD3+CD8+ | 40.9 | 3.329 | 27 | 1.8 | ** | |
CD4-CD8 ratio | 1.288 | 0.213 | 2.358 | 0.2358 | ** | Increased CD8+ (NS) |
% CD8+CD45RAhiCCR7+ (naive) | 20.56 | 2.774 | 50.3 | 2.354 | *** | Lower in BM |
% CD8+CD45RAloCCR7+ (CM) | 20.96 | 3.54 | 10.2 | 1.11 | * | Equivalent |
CD8+CD45RAloCCR7− (EM) | 16 | 3.196 | 17.3 | 1.571 | NS | Higher in BM |
% CD8+CD45RAhiCCR7− (EMR) | 19.2 | 2.589 | 20 | 1.16 | NS | Lower in BM |
% CD8+CD62L | 14.81 | 1.615 | 36.75 | 24.452 | *** | Higher in BM |
% CD8+27+28+ | 33.7 | 5.581 | 57.75 | 3.181 | * | Higher in BM |
% CD8+27−28+ | 17.1 | 2.313 | 7.55 | 1.157 | *** | Equivalent |
% CD8+27+28− | 10.19 | 1.187 | 18.8 | 1.862 | *** | Equivalent |
% CD8+27−28− | 30.45 | 4.818 | 23.45 | 3.226 | NS | Lower in BM |
% CD8+CXCR3+ | 2.16 | 0.67 | 0.775 | 0.165 | * | Lower in BM |
% CD8+CCR5+ | 31 | 3.746 | 25.13 | 1.586 | * | Higher in BM |
% CD8+CXCR6+ | 4.745 | 0.92 | 2.55 | 0.53 | NS | Higher in BM |
% FOXP3+CD4+ | 5.35 | 0.87 | 6.772 | 0.54 | NS | |
% EBV latent peptide–specific IFN-γ CD3+CD8+ | 0.01 | 0.1114 | 0.2 | 0.5901 | * | Equivalent |
% EBV lytic peptide–specific IFN-γ CD3+CD8+ | 0.605 | 0.461 | 1.64 | 0.984 | NS | Higher in BM |
% CMV peptide–specific IFN-γ CD3+CD8+ | 0.25 | 0.123 | 0.59 | 0.6653 | NS | Lower in BM |
EBV-DNA copies/106 cells | 0 (0-42) | 2.871 | 2685 (0-3614) | 251.9 | ** | Lower in BM |
Characteristic . | PBSC . | PB . | P . | Findings of Moss in BM vs PB . | ||
---|---|---|---|---|---|---|
Median (range) . | SE . | Median (range) . | SE . | |||
Age, y | 66 (22-70) | 3.713 | 53 (24-72) | 2.79 | NS | |
Sex, M/F | 11/5 | NA | 13/13 | NA | NS | |
Diagnosis | 7 myeloma, 8 lymphoma, 1 Ewing sarcoma | NA | Healthy | NA | NA | 80% primary lung cancer, 20% secondary lung cancer |
% CD3+CD8+ | 40.9 | 3.329 | 27 | 1.8 | ** | |
CD4-CD8 ratio | 1.288 | 0.213 | 2.358 | 0.2358 | ** | Increased CD8+ (NS) |
% CD8+CD45RAhiCCR7+ (naive) | 20.56 | 2.774 | 50.3 | 2.354 | *** | Lower in BM |
% CD8+CD45RAloCCR7+ (CM) | 20.96 | 3.54 | 10.2 | 1.11 | * | Equivalent |
CD8+CD45RAloCCR7− (EM) | 16 | 3.196 | 17.3 | 1.571 | NS | Higher in BM |
% CD8+CD45RAhiCCR7− (EMR) | 19.2 | 2.589 | 20 | 1.16 | NS | Lower in BM |
% CD8+CD62L | 14.81 | 1.615 | 36.75 | 24.452 | *** | Higher in BM |
% CD8+27+28+ | 33.7 | 5.581 | 57.75 | 3.181 | * | Higher in BM |
% CD8+27−28+ | 17.1 | 2.313 | 7.55 | 1.157 | *** | Equivalent |
% CD8+27+28− | 10.19 | 1.187 | 18.8 | 1.862 | *** | Equivalent |
% CD8+27−28− | 30.45 | 4.818 | 23.45 | 3.226 | NS | Lower in BM |
% CD8+CXCR3+ | 2.16 | 0.67 | 0.775 | 0.165 | * | Lower in BM |
% CD8+CCR5+ | 31 | 3.746 | 25.13 | 1.586 | * | Higher in BM |
% CD8+CXCR6+ | 4.745 | 0.92 | 2.55 | 0.53 | NS | Higher in BM |
% FOXP3+CD4+ | 5.35 | 0.87 | 6.772 | 0.54 | NS | |
% EBV latent peptide–specific IFN-γ CD3+CD8+ | 0.01 | 0.1114 | 0.2 | 0.5901 | * | Equivalent |
% EBV lytic peptide–specific IFN-γ CD3+CD8+ | 0.605 | 0.461 | 1.64 | 0.984 | NS | Higher in BM |
% CMV peptide–specific IFN-γ CD3+CD8+ | 0.25 | 0.123 | 0.59 | 0.6653 | NS | Lower in BM |
EBV-DNA copies/106 cells | 0 (0-42) | 2.871 | 2685 (0-3614) | 251.9 | ** | Lower in BM |
Herpesvirus peptide–specific IFN-γ producing CD8+ T cells were detected by intracellular cytokine staining by stimulating PB or PBSCs with appropriate HLA class I–restricted viral peptide as previously described.8 Intravenous cyclophosphamide (2 g/m2) and subcutaneous G-CSF (10 mcg/kg) were used for PBSC mobilization. Leukapheresis was performed using a Cobe Spectra Gambro BCT continuous flow cell separator.
PBSC indicates peripheral blood stem cells; PB, peripheral blood; BM, bone marrow; NS, not significant; NA, not applicable; IFN-γ, interferon-γ; CM, central memory; EM, effector memory; and EMR, effector memory revertant.
P = .05-.01;
P = .01-.001;
P < .001.
Authorship
Acknowledgments: Work in the laboratory is supported by the NHMRC, Cancer Council of Queensland, the Leukaemia Foundation, RISS, and Atlantic Philanthropies.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Maher K. Gandhi, Level I, CBCRC Bldg, Queensland Institute of Medical Research, Herston Rd, Brisbane, Queensland, 4006 Australia; e-mail: [email protected].
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