Hematopoietic Bias Drives Lymphopenia in Myeloproliferative Neoplasms

Background:

Myeloproliferative neoplasms (MPNs) are characterized by excessive myelopoiesis, leading to complications such as thrombosis, myelofibrosis and leukemia. Less well understood are the T cell exhaustion and immune defects responsible for increased risk of certain infections and solid tumor malignancies. Patients with MPNs also develop lymphopenia, contributing to elevated neutrophil-to-lymphocyte ratios (NLR), which are associated with disease-related complications.¹² Although MPNs are known to originate in a hematopoietic stem cell (HSC), defined by its capacity for both lymphoid and myeloid differentiation, the most common driver mutation JAK2V617F is rarely found in lymphocytes. This study aims to understand how JAK2V617F hematopoiesis affects lymphoid differentiation in MPNs.

Methods:

We used primary patient samples and murine models to define the effects of JAK2V617F on lymphoid differentiation in vitro and in vivo. Blood samples from patients were collected and processed. JAK2V617F mutation allele frequency (MAF) was measured by droplet digital PCR in hematopoietic subsets fractionated from peripheral blood (PB) using fluorescence-activated cell sorting (FACS).² Single-cell genotyping with feature barcoding was used to define the effects of JAK2V617F copy number on lymphoid differentiation. Sorted common lymphoid progenitors (CLPs) from patients were differentiated into T cells in vitro. The effects of JAK2V617F on lymphopoiesis were assessed in a mixed chimeric transplant model and a JAK2V617F transgenic model. Blood counts and patient data were extracted from the Silver MPN Center Research Data Repository. A linear mixed effects model was used to determine the effect of MPN duration on absolute lymphocyte count (ALC) in patients with JAK2V617F MPNs.

Results:

Patients with MPNs (n=757) lost 17 (±3) lymphocytes/μL per year, independent of age or treatment, with younger patients experiencing more dramatic losses. JAK2V617F alleles were depleted in T cells relative to HSC/MPPs, with depletion worsening over time, indicating a bias against lymphopoiesis. In vitro differentiation of CLPs from PV patients (n=16) into T cell precursors showed a significant decrease in JAK2V617F MAF of 19% (±13%) at later T cell stages, suggesting a selective disadvantage during T cell differentiation. In contrast, MAF was maintained or increased in erythroblasts derived from myeloid progenitors. Single-cell genotyping with surface protein feature barcoding demonstrated that bi-allelic JAK2V617F mutated cells were even more strongly excluded from lymphocytes than mono-allelic mutants (median log2 fold change -7.2, IQR -Inf to -5.4, and -4.4, IQR -5.1 to -2.3, respectively).

In vivo studies in murine models showed that Jak2V617F was depleted in T and B lymphocytes compared to Jak2WT controls (mean Jak2V617F chimerism depleted by 6% (±4) in T cells and 15% (±8) in B cells, n=12). JAK2V617F transgenic mice exhibited reduced peripheral lymphocyte counts and impaired lymphopoiesis, with stage-specific blocks in T and B cell development. RNA-seq of JAK2V617F CLPs revealed suppression of pathways critical for cell proliferation and Notch signaling.

Conclusion:

Our findings demonstrate that the JAK2V617F driver mutation enforces a hematopoietic bias against lymphopoiesis, explaining the rarity of JAK2V617F lymphocytes and the development of lymphopenia in MPN patients. Cells with bi-allelic JAK2 mutations are even more strongly depleted from lymphoid populations. Impaired lymphoid differentiation is a key feature of JAK2V617F hematopoiesis, contributing to the elevated NLR and related complications in MPNs. These insights offer a new understanding of MPN pathobiology and underscore the need for further research into immune deficits in MPNs.

1. Larsen MK, Skov V, Kjær L, et al. Neutrophil-to-lymphocyte ratio and all-cause mortality with and without myeloproliferative neoplasms-a Danish longitudinal study. Blood Cancer J. 2024;14(1):28.

2. Abu-Zeinah G, Di Giandomenico S, Choi D, et al. Hematopoietic fitness of JAK2V617F myeloproliferative neoplasms is linked to clinical outcome. Blood Adv. 2022;6(18):5477-5481.

Scandura:Incyte: Membership on an entity's Board of Directors or advisory committees; Constellation: Consultancy, Membership on an entity's Board of Directors or advisory committees; SDP Oncology: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Protagonist Therapeutics: Membership on an entity's Board of Directors or advisory committees; Morphic: Consultancy; Medpacto: Research Funding; Calico: Consultancy. Lehmann:AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; BeiGene: Honoraria; Astellas: Research Funding.