Impaired Response to Influenza Vaccination in AML Patients Post-Chemotherapy Associated with a Highly Atypical B-Cell Profile
Authors: Goswami Meghali , Prince Gabrielle T. , Biancotto Angelique , Moir Susan , Cheung Foo , Kotliarov Yuri , Dunham Kimberly , Chen Jinguo , Shi Rongye , Zhou Huizhi , Golding Hana , Tang Jingrong , Tsang John S. , Dickler Howard B. , Noonan Kimberly , Smith B. Douglas , Borrello Ivan M. , Hourigan Christopher S. 
Published: October 2019

Abstract
The immunotherapy of cancer is arguably the most promising therapy under development, and vaccination against cancer antigens is a promising strategy to stimulate adaptive immune responses against malignant clones. However, the ability of patients treated with chemotherapy to respond appropriately to immune challenge may be suboptimal. This study was designed to determine the functional capacity of the immune system in adult acute myeloid leukemia (AML) patients who have completed chemotherapy and are potential candidates for immunotherapy. We used the response to influenza vaccination as a surrogate for the health of the immune system in AML patients in a complete remission (CR) post-chemotherapy.
Ten adult AML patients in CR after completion of intensive chemotherapy were recruited to the clinical protocol J1293. They were on average 37 weeks post-treatment (range 4-148) when receiving the 2012-2013 inactivated seasonal influenza vaccine. Peripheral blood samples were collected at baseline and 30 days post-vaccination. Ten age and sex matched healthy donors (HD) served as baseline controls. Serological response to vaccination was assessed via microneutralization assays; multi-parameter flow cytometry was used to characterize lymphocyte subsets. ELISPOT assays were used to evaluate lymphocyte function, microarrays were used to assess gene expression, and deep sequencing of the B-cell receptor heavy chain (IGH) was performed to determine expansion and clonality of B-cells. Wilcoxon rank-sum tests were used to assess statistical significance.
Only 2/10 (20%) patients seroconverted (AML responders, or AML-R) with a four-fold increase or greater in influenza-specific antibody. One responder was 148 weeks post-treatment; the other had acute promyelocytic leukemia (APL) and was 4 weeks post-treatment. Deep immunophenotyping revealed no striking differences in T-cell compartments between AML at baseline and HD, indicating rapid T-cell recovery after chemotherapy. In contrast, we observed a highly atypical B-cell profile. AML non-responders (AML-NR) at day 0 versus HD had significantly reduced frequencies of mature IgA+ (4.5% vs. 11%) and IgG+ (2.4% vs. 5.9%) B-cells (as a percentage of CD19+CD20+). Further dissection exposed markedly higher frequencies of CD10+CD27- transitional B-cells (36% vs. 16%, p<0.005) and dramatically fewer memory (resting, tissue-like, activated) B-cells (5% vs. 25%, p<0.005) (as a percentage of CD19+CD20+) in AML-NR at day 0 versus HD. There were no significant changes in any B-cell population at day 30 over baseline in any patient. Examining frequencies of transitional, naive, and memory B-cells in AML patients at day 0 when ranked by time since treatment showed a decrease in transitional B-cells with a corresponding increase in naive B-cells over time but no concurrent increase in memory B-cell frequencies (Fig 1). These data suggest B-cell deficiencies of several types: loss of the memory B-cell compartment due to chemotherapy, a subsequent excess of transitional B-cells, and a lack of naive B-cell development into specific, class-switched effectors of the antibody response, which together likely lead to humoral immune incompetence.
Interestingly, functional T-cell assays revealed that of 5 evaluable patients, including the 2 AML-R and 3 AML-NR, 5/5 (100%) patients had an increase in influenza-specific cytokine production (1.24 - 4.40x higher on day 30 over baseline), suggesting a functional T-cell response even with deficient influenza-specific antibody production. Supervised clustering of microarray data identified many upregulated genes in AML-NR related to apoptosis, BCR, IL2, IL-4, IL-8, and IL-12 signaling pathways, indicative of developing B-cells. IGH sequencing demonstrated AML-NR had greater variability in CDR3 length than seen in HD, consistent with an antigen inexperienced B-cell repertoire.
These data suggest that while some aspects of cellular immunity recover comparatively quickly, the humoral immune system is incompletely reconstituted in the year following intensive cytotoxic chemotherapy for AML. Abnormal frequencies of transitional and memory B-cells may explain the poor response to vaccination often seen in patients after chemotherapy. Furthermore, the uncoupled recovery of B-cell and T-cell immune capacity observed here might have implications for the success of immunotherapies based on vaccination.
Figure 1. Figure 1.

Disclosures
Noonan: Celgene: Speakers Bureau. Borrello:Celgene: Research Funding.