Identification of cross‐reactive antibodies for the detection of lymphocytes, myeloid cells and haematopoietic precursors in the naked mole rat

Abstract The naked mole rat (Heterocephalus glaber, NMR) is a rodent with exceptional longevity, low rates of age‐related diseases and spontaneous carcinogenesis. The NMR represents an attractive animal model in longevity and cancer research, but there are no NMR‐specific antibodies available to study its immune system with respect to age‐ and cancer‐related questions. Substantial homology of major NMR immune cell markers with those of Guinea pig, human and, to a lesser extent, mouse and rat origin are implicated for the existence of immunological cross‐reactivity. We identified 10 antibodies recognising eight immunophenotypic markers expressed on the NMR's T and B lymphocytes, macrophages/monocytes and putative haematopoietic precursors and used them for an immunophenotyping of leukocyte subsets of peripheral blood, spleen and bone marrow samples. Overall, we found that the leukocyte composition of NMR peripheral blood is comparable to that of mice. Notably, the frequency of cytotoxic T cells was found to be lower in the NMR compared to corresponding mouse tissues and human blood. Antibodies used in the present paper are available either commercially or from the scientific community and will provide new opportunities for the NMR as a model system in ageing‐ and cancer‐related research areas.

. Antibodies recognising NMR surface antigens expressed in RLM11 mouse T cells RLM11 mouse T cells were transfected with vector NMR CD14, stained with mouse antibodies recognising human CD14, clones TM1 and M5E2 (shown as negative control) and analysed by flow cytometry. Representative results from one of two experiments are shown. Gating strategy is shown in the Supporting Information Figure S4. Supporting Information Figure S1B. Antibodies recognising NMR surface antigens expressed in RLM11 mouse T cells RLM11 mouse T cells were transfected with vectors encoding NMR CD8a and CD14. Cells were stained with primary antibodies recognising Guinea pig CD8a (clone CT6) and a-mouse IgG A546 secondary antibodies and analysed by flow cytometry. Representative results from one of two experiments are shown. Gating strategy is shown in the Supporting Information Figure S4. Supporting Information Figure S1C. Antibodies recognising NMR surface antigens expressed in RLM11 mouse T cells RLM11 mouse T cells were transfected with vectors encoding NMR CD8a and CD14. Cells were stained with antibodies recognising human CD8a (clone GN11/134D7, shown as negative control) and CD14 (clone TM1) and analysed by flow cytometry. Representative results from one of two experiments are shown. Gating strategy is shown in the Supporting Information Figure S4.  Figure S1D Antibodies recognising primary NMR cells Cells were isolated from NMR blood, spleen and bone marrow. Cells were stained with antibodies recognising Guinea pig CD8a, human CD14, CD20, CD45R/B220 and CD120b/TNFR2 and analysed by flow cytometry. Clone B607 recognising Guinea pig CD8a and antibodies, recognising human CD14, CD20, CD45R/B220 and CD120b/TNFR2 are shown as negative control. a-Mouse IgG A546 secondary antibodies were used with antibodies against CD8a. Representative results from one of two experiments with a total of two NMR samples are shown. Gating strategy is shown in the Supporting Information Figure S4. Unstained a-rat IgG A647

GFP
CD3-12 + a-rat IgG A647 Supporting Information Figure S2. Antibodies recognising the intracellular domain of NMR CD3e A. HEK 293T human cells were transfected with vectors encoding NMR CD3e and CD4 (used as negative control). Representative results from one of two experiments are shown. B. Splenocytes isolated from NMR. A and B. Cells were fixed, permeabilised and stained with rat CD3-12 (A and B) and mouse PC3/188a (A only) antibodies recognising human CD3e. a-Rat IgG A647 and a-mouse IgG A546 secondary antibodies were used respectively. Cells were analysed after exclusion of debris, non-lysed erythrocytes, aggregated and dead cells (Supporting Information Figure S1). B. Representative results from one of two experiments with a total of two NMR samples are shown. (A and B) Gating strategy is shown in the Supporting Information Figure S4.   Supporting Information Figure S3. Antibodies recognising NMR MHC-II (example of Screening strategy II). A. Cells were isolated from NMR blood and spleen, stained with antibodies, recognising Guinea pig MHC-II (clones 27E7, MSgp8 and 25E3 [used as negative control]) and secondary a-mouse IgG A546 antibodies. Cells were analysed by flow cytometry after exclusion of debris, non-lysed erythrocytes, aggregated and dead cells (gating strategy is shown in the Supporting Information Figure S4). B. Cells, positive and negative for staining by antibodies against Guinea pig MHC-II clones 27E7 and MSgp8, were isolated by FACS from NMR bone marrow and analysed by Q-RT-PCR for expression of mRNA encoding NMR MHC-II antigen. PCR was set up in triplicates. NMR b-actin (a) and GAPDH (b) were used as housekeeping genes for data normalisation. Supporting Information Figure S4. Gating strategy for conventional flow cytometry. Cells were isolated from NMR spleen, blood and bone marrow. Viability Dye eFluor ® 780 was used for exclusion of dead cells.
Representative results from one of five independent experiments with a total of six NMR samples are shown.

SSC-
Supporting Information Figure S5. Identification of putative B cells (complete version of Figure 1C) Cells were isolated from NMR spleen, blood and bone marrow and stained with antibodies recognising Guinea pig IgM (clone 31D2, conjugated with PE) and MHC-II (clone MSgp8, conjugated with PacB ) ( Table 1). G1 -G4 sub-population ( Figure 1A) were analysed separately after exclusion of debris, non-lysed erythrocytes, aggregated and dead cells (gating strategy is shown in the Supporting Information Figure S4). Representative results from one of three experiments with a total of three NMR samples are shown.
Supporting Information Figure S6. Gating strategy for imaging flow cytometry. Cells were isolated from NMR spleen, blood and bone marrow and stained with antibodies recognising Guinea pig MHC-II (clone MSgp8, conjugated with PacB) and IgM (clone 31D2, conjugated with PE) and human CD14 (clone TM1, conjugated with Cy5) (

Bone marrow
Supporting Information Figure S7A. Imaging flow cytometry analysis of NMR immune cells. Putative B cells. Cells were isolated from NMR spleen, blood and bone marrow and stained with antibodies recognising Guinea pig MHC-II (clone MSgp8, conjugated with PacB) and IgM (clone 31D2, conjugated with PE) and human CD14 (clone TM1, conjugated with Cy5) ( Table 1). Images of IgM + MHC-II + CD14cells are shown after exclusion of debris, non-lysed erythrocytes, aggregated and dead cells (see the Supporting Information Figure S6 for gating strategy). Representative results from one of two experiments with a total of two NMR samples. n.s. -non-specific autofluorescence.

Spleen
Bone marrow Supporting Information Figure S7B. Imaging flow cytometry analysis of NMR immune cells. Putative monocytes/macrophages. Cells were isolated from NMR spleen, blood and bone marrow and stained with antibodies recognising Guinea pig MHC-II (clone MSgp8, conjugated with PacB) and IgM (clone 31D2, conjugated with PE) and human CD14 (clone TM1, conjugated with Cy5) ( Table 1). Images of IgM -MHC-II + CD14 + cells are shown after exclusion of debris, non-lysed erythrocytes, aggregated and dead cells (see the Supporting Information Figure S6 for gating strategy). Representative results from one of two experiments with a total of two NMR samples. n.s. -non-specific autofluorescence.

Bone marrow
Supporting Information Figure S7C. Imaging flow cytometry analysis of NMR immune cells. CD14 + subset of putative granulocytes. Cells were isolated from NMR spleen, blood and bone marrow and stained with antibodies recognising Guinea pig MHC-II (clone MSgp8, conjugated with PacB) and IgM (clone 31D2, conjugated with PE) and human CD14 (clone TM1, conjugated with Cy5) ( Table 1). Images of IgM -MHC-II -CD14 + cells are shown after exclusion of debris, non-lysed erythrocytes, aggregated and dead cells (see the Supporting Information Figure S6 for gating strategy). Representative results from one of two experiments with a total of two NMR samples. n.s. -non-specific autofluorescence.  Figure S8. Identification of putative T cells (complete version of Figure 1E). Cells were isolated from NMR spleen, blood and bone marrow, pre-stained with antibodies, recognising Guinea pig CD8a (clone CT6, used with a-mouse IgG A546 secondary antibody) or mouse CXCR3 (clone CXCR3-173, conjugated with PE), fixed and stained with anti-CD3e antibodies (clone CD3-12, used with a-rat IgG A647 secondary antibody). G1 (FSC-A-low/SSC-A-low) sub-population ( Figure 1A), representing lymphocytes, was analysed after exclusion of debris, non-lysed erythrocytes, aggregated and dead cells (gating strategy is shown in the Supporting Information Figure S4). Representative results from one of three experiments with a total of three NMR samples are shown.

MHC-II-PacB
Bone marrow Supporting Information Figure S9. Identification of putative monocytes/macrophages (complete version of Figure 1F). Cells were isolated from NMR spleen, blood and bone marrow, stained with antibodies recognising Guinea pig MHC-II (clone MSgp8, conjugated with PacB) and human CD14 (clone TM1, conjugated with Cy5) ( Table 1) and analysed by conventional flow cytometry. G1-G4 sub-population ( Figure 1A) were analysed separately after exclusion of debris, non-lysed erythrocytes, aggregated and dead cells (gating strategy is shown in the Supporting Information Figure S4). Representative results from one of three experiments with a total of three NMR samples are shown.

aCD34-APC/a-c-Kit-PE-Cy5
Supporting Information Figure S10A. Analysis of putative haematological precursors (complete version of Figure 1G). Cells were isolated from NMR spleen, blood and bone marrow and stained with antibodies recognising human CD34 (clone AC136, conjugated with APC) and mouse c-Kit/CD117 (clone ACK4, conjugated with PE-Cy5) ( Table 1). G1 -G4 sub-populations ( Figure 1A) were analysed separately after exclusion of debris, non-lysed erythrocytes, aggregated and dead cells (gating strategy is shown in the Supporting Information Figure S4). Representative results from one of three experiments with a total of three NMR samples are shown. a-c-Kit-PE-Cy5 Supporting Information Figure S10B. Analysis of putative haematological precursors (individual stainings -control of channels compensation). Cells were isolated from NMR bone marrow and stained with antibodies recognising human CD34 (clone AC136, conjugated with APC) or mouse c-Kit/CD117 (clone ACK4, conjugated with PE-Cy5) ( Table 1). G1 -G4 sub-populations ( Figure 1A) were analysed separately after exclusion of debris, non-lysed erythrocytes, aggregated and dead cells (gating strategy is shown in the Supporting Information Figure S4). Representative results from one of three experiments with a total of three NMR samples are shown.