CD4⁺ T Cells Coexpressing CD134 (OX40) Harbor Significantly Increased Levels of Human Herpesvirus 6B DNA Following Umbilical Cord Blood Transplantation

Abstract

Human herpesvirus 6B (HHV-6B) commonly reactivates after umbilical cord blood transplantation (UCBT) and is associated with delayed engraftment, fever, rash, and central nervous system dysfunction. Recently, CD134 (OX40) has been implicated as a potential viral entry receptor. We evaluated CD4⁺CD134⁺/^neg-lo and CD8⁺CD134⁺/^neg-lo cells at day 28 after UCBT in 20 subjects with previously documented HHV-6 reactivation and persistent viremia. Analysis of CD4⁺CD134⁺ cells as compared to CD4⁺CD134^neg-lo cells showed 0.308 versus 0.129 copies of HHV-6B/cell (P = .0002). CD8⁺CD134^+/neg-lo cells contained little to no HHV-6B copies. Following UCBT, CD4⁺CD134⁺ cells harbor significantly increased levels of HHV-6B, suggesting that CD134 (OX40) may facilitate viral entry.

Human herpesvirus 6 (HHV-6) infects >90% of the general population by age 2 years [1], establishes lifelong persistence in a latent form, and reactivates during periods of T-cell dysfunction. Two species of HHV-6 exist (A and B), but HHV-6B is the predominant species that reactivates following allogeneic hematopoietic cell transplantation (allo-HCT) [1]. HHV-6B reactivation occurs commonly after allo-HCT and is associated with end organ disease, including pneumonitis, fever, rash, central nervous system dysfunction, delayed engraftment (or graft failure), graft–versus-host disease (GvHD), and cytomegalovirus (CMV) reactivation [2]. The incidence of HHV-6B reactivation or viremia after transplantation ranges from 30% to 71%, which varies significantly, depending on stem cell source [3]. Many patients will have viremia without evidence of end organ disease and can remain viremic for months to years after allo-HCT [4].

There is limited understanding regarding the interactions between HHV-6B and the immune system following allo-HCT. Epidemiologic studies have clearly demonstrated that HHV-6B reactivation occurs more frequently after umbilical cord blood transplantation (UCBT) as compared to peripheral blood or bone marrow stem cell transplantations [3]. The cause of this discrepancy remains unclear. One study evaluated the relationship between expression of CD46 (the primary cellular receptor for HHV-6A and proposed secondary receptor for HHV-6B) between UCB, peripheral blood, and bone marrow stem cell sources. In this study, the differences in CD46 expression did not account for the increased frequency of HHV-6B reactivation after UCBT [5].

Recently, CD134 (OX40) was identified as a primary receptor molecule for HHV-6B [6]. CD134 is a member of the tumor necrosis factor receptor superfamily. Expression of this receptor is found on activated CD4⁺ T lymphocytes and, to a lesser degree, CD8⁺ T lymphocytes [7]. Interestingly, the expression of CD134 on peripheral CD4⁺ and CD8⁺ T cells also correlates with the development of acute and chronic GvHD [8, 9].

Because CD134 expression may be used for HHV-6B cellular entry, we hypothesized that CD4⁺CD134⁺ T cells may serve as a reservoir for HHV-6B reactivation in patients after allo-HCT. Given the high rates of HHV-6B reactivation following UCBT, we performed a comparative analysis of viral copy numbers among CD134^+/neg-lo T lymphocytes in a cohort of 20 subjects with persistent viremia following UCBT. Identification of a viral reservoir could be crucial in our understanding of HHV-6B immune biology and, possibly, lead to identification of novel targets for future therapeutic interventions.

METHODS

Subject and Sample Characteristics

Twenty subjects with leukemia or lymphoma who underwent UCBT during 2011–2015 at our institution and experienced HHV-6 reactivation (previously diagnosed on the basis of non–species-specific HHV-6 polymerase chain reaction [PCR] findings in whole blood) after UCBT were identified. Details of the transplantation procedures (ie, the conditioning regimen and GvHD prophylaxis) have previously been published [10]. Samples of viable peripheral blood mononuclear cells were obtained from subjects a median of 28 days after transplantation (range, 20–32 days) and stored in liquid nitrogen. Flow cytometry was used to sort cells into 4 groups based on T-cell phenotype (CD4⁺CD134⁺, CD4⁺CD134^neg-lo, CD8⁺CD134⁺, and CD8⁺CD134^neg-lo). Total nucleic acid was extracted from the resulting 80 samples for HHV-6A and HHV-6B quantification. A reference gene, RPP30, was used to normalize the data and control for numbers of cells collected.

This study was approved by the Committee on the Use of Human Subjects in Research at the University of Minnesota. All samples were collected from patients after informed written consent was obtained from the parents or guardians on behalf of the child participants.

Fluorescence-Activated Cell-Sorting (FACS)–Based Purification

The following antibodies were used for FACS-based purification of lymphocyte populations:

anti-CD134 (OX40) PE (catalog no. 350004; BioLegend), anti-CD8 FITC (catalog no. 555634; BD Pharmingen), anti-CD3 PerCP-Cy5.5 (catalog no. 300430; BioLegend), and anti-CD4 AlexaFluor 700 (catalog no. 555349; Pharmingen). Discrimination between live and dead cells was performed using the Live Dead Fixable Aqua Dead Cell Stain Kit from Molecular Probes (catalog no. L34966). Cell sorting was performed on a BD FACSAria II sorter. Discrimination between CD134⁺ cells and CD134⁻ cells was based on the top quartile (CD134⁺) and bottom quartile (CD134^neg-lo) of CD134 expression. Samples were directly sorted into 3.5 mL of RLT buffer (Invitrogen). The median proportion ( ± SD) of CD4⁺CD134^neg-lo, CD4⁺CD134⁺, CD8⁺CD134^neg-lo, and CD8⁺CD134⁺ cells among live lymphocyte–gated cells was 2.2% ± 3.3%, 3.1% ± 4.1%, 1.0% ± 4.7%, and 0.7% ± 1.8%, respectively.

HHV-6 Analysis

HHV-6 quantitative testing was performed by the diagnostic laboratory division of Coppe Healthcare Solutions (Waukesha, WI). Total nucleic acid from each sample was purified using the QIAamp MinElute Virus Spin kit (Qiagen) and concentrated to 50 µL (7-fold), with 5 µL of sample used in each PCR assay. Documentation of replicating virus was determined by reverse-transcription PCR analysis for the presence of HHV-6A and HHV-6B species common late-response and species-specific immediate early (IE) gene transcripts. The messenger RNA transcript of the gene encoding human ornithine decarboxylase 1 was used to control for the quality of extraction; the limit of detection for these assays is one HHV-6A or HHV-6B actively infected cell per 1000 mononuclear cells. Quantitative real-time PCR (qPCR) targeting the IE genes of HHV-6A and HHV-6B DNA was also performed on each sample. Total nucleic acid was purified as described above, and an internal ribonuclease reference gene (RPP30) was used for cell count normalization and to evaluate for inherited chromosomally integrated HHV-6 (ciHHV-6). The limit of detection for these assays is 1 copy of viral DNA per 1000 nucleated cells. Cell counts in the 80 samples analyzed range from 8 to 49 745, as determined by FACS analysis. All CD4⁺ samples contained sufficient DNA and cell counts to estimate the number of viral DNA copies per cell.

Statistical Analysis

A paired t test was performed (via Prism software version 7.0a for Mac OS X) to directly compare the number of HHV-6B copy numbers among CD4⁺CD134⁺ and CD4⁺CD134^neg-lo T-cell populations separated from each subject, as outlined above.

RESULTS

Clinical Data and HHV-6 Viremia

Subject demographic characteristics are shown in Table 1. Non–species-specific HHV-6 viremia had been previously documented in all subjects a median of 17 days after transplantation (range, 9–380 days), with persistently elevated viremia levels of >3000 copies/mL in 14 subjects and >25 000 copies/mL in 6 subjects for a median duration of 4 months (range, 1–11 months; data not shown). The median peak viremia level was 160 000 copies/mL (range, 5500–2 × 10⁶ copies/mL). Eighteen of 20 subjects had engraftment of >90% of donor cells at the time of sample collection (with values of 84% and 27% for the remaining 2).

Table 1.

Demographic, Clinical, and Transplantation Characteristics of Subjects and Data on Human Herpesvirus 6B (HHV-6B)

Subject	Sex	Age, y	HCT	Conditioning	Posttransplantion Day of Sample Collection	Time in Days to		GvHD Status		Survival Status	HHV-6B Load, Copies, No.
						ANC of > 500 Neutrophils/μL	PLT Count of >2×104 Platelets/μL	Acute, Grade 2–4	Chronic		Per CD4+ CD134+ Cell	Per CD4+ CD134neg-lo Cell
1a	M	49	dUCB	MA	28	20	46	Yes	Yes	Dead	2.74b	1.625b
2	F	11	sUCB	MA	24c	16	38	No	No	Dead	0.076	0
3	M	63	dUCB	NMA	28	28	56	Yes	No	Dead	0.067d	0.134d
4	F	63	dUCB	NMA	20	15	31	No	No	Alive	0.591	0.12
5	M	6	sUCB	MA	25	15	66	No	No	Alive	0.081	0.023
6	M	25	dUCB	MA	28	26	…	No	No	Alive	0.119	0
7	F	44	dUCB	NMA	28	6	23	No	No	Alive	0.104	0.037
8	M	1	dUCB	MA	28	13	30	No	No	Alive	0.055	0.004
9	M	62	dUCB	NMA	27	19	55	Yes	No	Alive	1.147e	0.953e
10a	F	8	sUCB	MA	…f	GF	GF	No	No	Dead	…g	…g
11a	F	11	sUCB	MA	29	13	105	No	No	Alive	0h	0h
12	F	34	dUCB	MA	27	28	49	Yes	Yes	Alive	0.49	0.181
13	F	31	dUCB	MA	32	6	31	Yes	No	Dead	0.081	0.059
14	M	58	dUCB	NMA	28	10	168	No	No	Dead	0.635	0.316
15	F	32	sUCB	MA	22	26	41	Yes	No	Alive	0.427	0.073
16	M	9	sUCB	NMA	22c	0	36	Yes	No	Alive	0.167	0
17	M	8	sUCB	MA	28	21	40	No	No	Alive	0.134	0.006
18a	F	27	dUCB	MA	28	35	195	Yes	No	Alive	0h	0h
19	F	9	sUCB	MA	28	16	41	Yes	No	Alive	0.506	0.159
20	M	19	dUCB	MA	29	24	56	No	Yes	Alive	0.241	0

Abbreviations: ANC, absolute neutrophil count; dUCB, double umbilical cord blood; GF, graft failure; GvHD, graft-versus-host disease; HCT, hematopoietic cell transplantation; MA, myeloablative; NMA, nonmyeloablative; PLT, platelet; sUCB, single umbilical cord blood.

^a Subjects were not included in statistical analyses.

^b CD8⁺ T cells were also found to have >1 copy of HHV-6B/cell (data not shown). Subject has converted to chromosomally integrated HHV-6B positivity following transplantation with donor chromosomally integrated HHV-6B–positive cells.

^c Sample was collected after second umbilical cord blood transplantation.

^d Only subject in cohort with greater HHV-6B copies in CD4⁺CD134^neg-lo cells than CD4⁺CD134⁺ cells.

^e Although approximately 1 copy of HHV-6B/cell was observed in CD4⁺ T cells, CD8⁺ T cells were found to be HHV-6B negative, thus ruling out chromosomally integrated HHV-6B.

^f Sample was collected after third umbilical cord blood transplantation.

^g HHV-6A–positive only, with no HHV-6B detected in any T-cell population.

^h No virus was detected in any T-cell population.

Identification of HHV-6B

HHV-6B was identified in T-cell populations in 17 of 20 subjects (Table 1). In 1 subject (subject 10), HHV-6A was identified, and 2 subjects (subjects 11 and 18) were negative for either HHV-6A or HHV-6B. Subject 1 was found to have ciHHV-6B. Thus, 4 subjects were excluded from the statistical analysis, of whom 2 were negative for HHV-6A or HHV-6B (subjects 11 and 18), and 1 subject each had HHV-6A (subject 10) and ci-HHV-6B (subject 1) identified.

CD4⁺ T Cells and HHV-6B

A direct comparison of HHV-6B copy numbers from CD4⁺CD134⁺ T cells to copy numbers from CD4⁺CD134^neg-lo T cells for each subject is provided in Figure 1A. The HHV-6B copy number was significantly greater in CD4⁺CD134⁺ T cells as compared to CD4⁺CD134^neg-lo T cells, with a mean of 0.308 copies/cell versus 0.129 copies/cell (n = 16; P = .0002; Figure 1B).

Figure 1.

Human herpesvirus 6B (HHV-6B) copies per CD4⁺CD134⁺ versus CD4⁺CD134^neg-lo cells. A, Direct comparison of HHV-6B copies/cell for each subject, with the number of HHV-6B copies per CD4⁺CD134^neg-lo cell shown in red and the number of copies per CD4⁺CD134⁺ cell shown in blue. B, Comparison of the mean HHV-6B copy numbers. Data are mean values ± standard errors of the mean. Statistical analysis performed via a paired Student t test yielded a P value of .0002.

In 12 subjects, HHV-6B copies were detected in both CD134⁺ and CD134^neg-lo populations, with 5.2-fold greater HHV-6B levels in the CD134⁺ cells as compared to the CD134^neg-lo CD4⁺ T cells. In 4 subjects (subjects 2, 6, 16, and 20), HHV-6B DNA was detected in CD4⁺CD134⁺ T cells but not in CD4⁺CD134^neg-lo T cells. There were no instances in which subjects with CD4⁺CD134^neg-lo T cells containing HHV-6B had CD134⁺ cells that did not contain HHV-6B. There was 1 case (subject 3) in which CD4⁺CD134^neg-lo T cells contained more copies of HHV-6B than CD4⁺CD134⁺ T cells. Of note, subject 9 had an HHV-6B copy number >1.0 copy/cell in the CD4⁺CD134⁺ population; however, viral DNA was absent in CD8⁺ T cells, thus ruling out ciHHV-6B.

CD8⁺ T Cells and HHV-6B

CD8⁺ T cells are infected much less efficiently by HHV-6B, compared with CD4⁺ T cells [11]. As anticipated, a minimal level of HHV-6B DNA was identified in only 2 of 16 subjects with HHV-6B identified in corresponding CD4⁺ T cells (minimal detection level, <0.01 copies/cell; data not shown). Subject 1 was engrafted with ciHHV-6B from donor cells, and therefore the CD8⁺ T cells contained ≥1 copy of HHV-6B in every cell regardless of CD134 expression.

DISCUSSION

In a cohort of subjects who underwent UCBT and experienced HHV-6B reactivation, we show that CD4⁺ T cells coexpressing CD134 (OX40) contain significantly greater numbers of HHV-6B copies than CD4⁺ T cells without CD134 expression. This work builds on that of Tang et al, who recently described CD134 as a primary receptor molecule for HHV-6B infection in vitro [6].

We propose 3 potential mechanisms to explain these results. First, CD134 may directly promote enhanced cellular entry for HHV-6B. Our findings suggest that coexpression of the CD134 receptor does not appear to be exclusively required for HHV-6B cellular entry in vivo, as CD4⁺ T cells without CD134 coexpression were also found to be harboring HHV-6B (albeit at significantly lower levels than CD134⁺ populations). Second, as CD134 is known to be present only on activated T lymphocytes, this may represent a coincidental marker of T cells that are inherently capable of increased intracellular activity overall, likely including intracellular viral replication. Finally, surface CD134 receptor expression may be a consequence of intracellular viral reactivation/replication. However, this seems less likely because Tang et al have demonstrated that productive HHV-6B infection of T cells generally results in downregulation of surface CD134 in vitro [6].

There are many potential clinical implications of CD134⁺ cells harboring increased levels of HHV-6B early after UCBT. CD134 expression on peripheral CD4⁺ and CD8⁺ T cells has been strongly correlated with acute and chronic GvHD in patients undergoing allogeneic stem cell transplantation [8, 9], and there is mounting evidence of an association between HHV-6B reactivation and development of acute GvHD [12]. This possible link warrants further investigation.

Several authors have highlighted the importance of distinguishing between HHV-6B reactivation and ciHHV-6B in patients exhibiting excessive HHV-6B viremia levels after transplantation [13]. In our study, we identified 2 subjects (subjects 1 and 9) with samples containing ≥1 copy of HHV-6B per CD4⁺CD134⁺ cell. In subject 1, additional analysis revealed ≥1 copy of HHV-6B in both CD4⁺ and CD8⁺ T cells, with confirmed 100% donor engraftment of T cells at the time of sample collection. Taken together, it is reasonable to conclude that this subject's excessive HHV-6B viremia level can be explained by likely conversion to ciHHV-6B, a phenomenon that has been repeatedly documented [14]. In contrast, subject 9 from our cohort demonstrated unusually high HHV-6B copy numbers in CD4⁺ T cells but no virus in CD8⁺ T cells, suggesting HHV-6B reactivation in this subject, rather than ciHHV-6B. Regardless, emerging evidence suggests the possibility of clinical sequelae attributable to reactivation of ciHHV-6B [15], and subsequent management remains an area of current debate.

It is estimated that 30%–71% of all patients who undergo UCBT will experience HHV-6B reactivation after transplantation [3], which is associated with several transplantation-related morbidities, such as encephalitis, delayed engraftment/graft failure, fever with rash, pneumonitis, hepatitis, and GvHD [2]. In this pilot study of 20 subjects, we acknowledge that further studies are needed to confirm our results and expand on the data from this investigation. Our population was unique in that it comprised a cohort of homogenously treated subjects who underwent UCBT and had documented high-level and prolonged viremia. Of note, assayed samples were collected at a relatively early time point. Regardless, a greater understanding of CD134 expression after transplantation may help account for the increased prevalence of HHV-6B reactivation associated with UCBT versus other graft sources and could potentially lead to identification of novel targets for immunotherapy (such as use of soluble decoy receptors) to prevent or mitigate HHV-6–related complications after transplantation.

In summary, our findings indicate that, early (approximately 28 days) after UCBT, CD4⁺ T cells coexpressing CD134 (OX40) contain increased copies of HHV-6B. This is the first evidence of a potential CD134⁺ cellular reservoir within the context of HHV-6B reactivation after transplantation. Alternatively, CD134 induction may be a direct consequence of viral reactivation and productive infection or may serve as a marker of activated T cells capable of increased viral replication. Further characterization is necessary to enhance our current understanding of the mechanism of HHV-6B reactivation after transplantation and may uncover new targets for exploitation of future therapies aimed at minimizing adverse outcomes related to HHV-6B reactivation in transplant recipients.