Tasseomancy, the art of divining the future from tea leaves, developed in Europe only shortly after tea merchants introduced tea from China. Similarly, immunophenotyping of bronchoalveolar lavage (BAL) cells from lung transplant recipients has been interpreted for nearly as long as lung transplantation has been performed. While its clinical utility and limitations are still being defined, unlike tea leaf interpretation, BAL immunophenotyping has potential to improve lung transplant diagnosis. It has been regarded as an attractive adjunct in the diagnosis and differentiation of acute lung allograft rejection and infection. These conditions can be clinically challenging because of overlapping or absent symptoms and radiological signs, low levels of agreement on pathological findings, and challenges in the interpretation of microbiological studies.1,2 Thus, diagnoses are made via integration of multiple studies each with poor test characteristics. As technology advances, clinicians are frequently presented with more data but paradoxically less information.
Outwardly, BAL immunophenotyping could arbitrate ambiguous diagnoses as it provides tissue-level context for clinical decisions. A 4-factor scoring system incorporating BAL percentages of monocytes and lymphocytes among leukocytes, and percentages of CD25+ and natural killer (NK) cells among lymphocytes had fair performance in differentiating infection from rejection and identified a subset of patients with otherwise unremarkable biopsy results who developed chronic lung allograft dysfunction (CLAD).3 However, for each of these parameters, there was wide overlap in confidence intervals between infection and rejection. In Bayesian terms, BAL immunophenotyping parameters, including the total NK cell percentage, have a small but detectable impact on the posttest probability of disease. At the same time, while NK cells are increasingly recognized as mediators of important lung transplant outcomes, their interpretation is complicated by a myriad of potential roles and phenotypes.4 More granular receptor phenotyping may be one method to improve the power of flow cytometry in clinical diagnosis.
In this edition of Transplantation, Sullivan et al describe one such analysis of an NK cell subset expressing the CD94-NKG2C receptor complex, which recognizes cytomegalovirus (CMV)-infected host cells with memory-like capabilities (Figure 1).5,6 CMV remains a significant cause of morbidity and mortality in lung allograft recipients, and NK cells expressing the CD94-NKG2C receptor have been associated in bone marrow transplant cohorts with improved CMV control.7,8 The current study demonstrates that lung transplant recipients at increased risk for CMV disease, based on donor and recipient serologies, have higher proportions of NKG2C+ NK cells in BAL that were largely mirrored in synchronous peripheral blood samples.6 NKG2C+ NK cells also had an activated phenotype relative to NKG2C- NK cells. One important caveat is that CMV donor and recipient seropositive subjects were excluded from study. Strikingly, in many recipients, the observed increase in NKG2C+ NK cells followed the withdrawal of CMV antiviral prophylaxis as did the detection of CMV in BAL or plasma. These findings demonstrate an association between activated NKG2C+ NK cells and CMV infection in lung transplant recipients.
The major unanswered question is how to interpret the presence of NKC2C+ NK cells in lung allograft recipients’ BAL. It may be that strong CMV immunity is protective against invasive disease and thus elevated measures of this immunity could be grounds for reducing antiviral prophylaxis. However, the authors found no correlation between BAL NKC2C+ NK cell percentage and peripheral blood CMV viral load. Our group recently described increased proportions of NKG2C+ NK cells in BAL preceding CMV viremia, and no differences in NKG2C+ NK cells between recipients with single or multiple occurrences of CMV viremia.9 Further, lung allograft recipients with higher median proportions of BAL NKG2C+ NK cells were >4 times as likely to develop CLAD or death compared with the lower median group. Together, these findings would raise concern that premature withdrawal of CMV antiviral prophylaxis based on BAL NKG2C+ NK cell percentage could, in fact, put recipients at increased risk for further viremia or CLAD. Given the observed cytotoxic phenotype of CMV-specific NK cells, the effect of these cells in blunting CMV viremia could come at the cost of greater allograft injury.
How does a clinician incorporate these findings into clinical practice? With respect to the assessment of CMV infection, BAL NKG2C+ NK cell percentage appears to reflect the CMV allograft burden. Nonetheless, challenges in standardization of BAL protocols between centers limit their generalizability.10 While it is suspected that increased percentages of NKG2C+ NK cells in BAL might identify patients who could benefit from longer duration antiviral prophylaxis, further study is needed. In recommending therapies for lung allograft infection and rejection more generally, BAL immunophenotyping results generate low-information likelihood ratios. As Thomas Bayes described in 1763, the probability of an event can be calculated as the product of its prior probability and the test likelihood ratio. Thus, while more informative than reading tea leaves, BAL immunophenotyping results continue to require strong consideration of clinical context.
The author thanks John R. Greenland and Blake Charlton for their input.
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3. Greenland JR, Jewell NP, Gottschall M, et al. Bronchoalveolar lavage cell immunophenotyping facilitates diagnosis of lung allograft rejection. Am J Transplant. 2014;14:831–840.
4. Calabrese DR, Lanier LL, Greenland JR. Natural killer cells in lung transplantation. Thorax. [Epub ahead of print. October 31, 2018]. doi: 10.1136/thoraxjnl-2018-212345.
5. Foley B, Cooley S, Verneris MR, et al. Human cytomegalovirus (CMV)-induced memory-like NKG2C(+) NK cells are transplantable and expand in vivo in response to recipient CMV antigen. J Immunol. 2012;189:5082–5088.
6. Sullivan LC, Harpur CM, Stankovic S, et al. Enrichment of cytomegalovirus-induced NKG2C+ natural killer cells in the lung allograft. Transplantation. 2019;103:1689–1699.
7. Paraskeva M, Bailey M, Levvey BJ, et al. Cytomegalovirus replication within the lung allograft is associated with bronchiolitis obliterans syndrome. Am J Transplant. 2011;11:2190–2196.
8. Foley B, Cooley S, Verneris MR, et al. Cytomegalovirus reactivation after allogeneic transplantation promotes a lasting increase in educated NKG2C+ natural killer cells with potent function. Blood. 2012;119:2665–2674.
9. Calabrese DR, Chong T, Wang A, et al. NKG2C natural killer cells in bronchoalveolar lavage are associated with cytomegalovirus viremia and poor outcomes in lung allograft recipients. Transplantation. [Epub ahead of print. September 11, 2018]. doi: 10.1097/TP.0000000000002450.
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