Thymic stroma is affected due to the lack of VAMP8

A constant bidirectional crosstalk between the thymic stroma and the developing T lymphocytes is critical for the survival of both. Reduced thymus cellularity in the VAMP8 -/^- small and sick mice could be due to a defect of the thymocyte precursors to proliferate or in the thymic stromal cells that fail to signal the developing thymocytes. In order to

Chapter 3. Results

transplanted the bone marrow derived stem cell of VAMP8 ^-/^- small and sick and VAMP8⁺/^- control mice into RAG2^-/^- γc^-/^- recipient mice. The hematopoetic stem cells have a potential to differentiate into T- as well as B-lymphocytes when subjected to the right environment and stimulus.

The RAG2^-/^- γc^-/^- mice inturn, have a fully functional thymic stroma capable of providing the right microenviroment for the development of the T-lymphocytes. However, these mice do not have any functional T and B lymphocytes because Rag2 (recombinase activating enzyme 2) encodes the DNA recombinase enzyme required for VDJ recombination of the B-cell receptor and T-cell receptor gene loci and thereby the production of BCR and TCR proteins. Additionally, the common gamma chain (γc) is a subunit of the cytokine receptors for IL-2, -4, -7, -9, and -15 on the lymphocytes. The γc can combine with other ligand-specific receptors to direct lymphocytes to respond to different cytokines. Therefore, lymphocyte development is greatly compromised in the γc^-/^- mice. These mice lack natural killer (NK) cells and produce very few T and B lymphocytes. The double knock out mice for RAG2 and γc therefore completely lack T and B lymphocytes and NK cells. These mice were obtained from Professor Dr. Rodewald, from the Institite of Immunology, University of Ulm and kept in the C57BL/6J x C57BL/10^SbSnAi background.

In three independent experiments, bone marrow derived from 10-12 day old small and sick VAMP8^-/^- mice and VAMP8⁺/^- littermates were transplanted into irradiated RAG2^-/^- γc^-/ -mice. After 4 weeks, 7 weeks and 9 weeks of transplantation, blood was collected from the recipient RAG2^-/^- γc^-/^- mice and lymphocytes were stained with antibodies against CD4, CD8, CD3 to visualize T-lymphocytes, B220 and CD19 for the B-lymphocytes and NK1.1 for NK cells. Parallel samples were stained with isotype controls.

After 4 weeks of transplantation, there were B220 positive (B220⁺CD19⁺) B-lymphocytes in all the RAG2 ^-/^- γc^-/^- recipients (Figure 3.31). However, there were no T lymphocytes in the RAG2 ^-/^- γc^-/^- recipients of both the test and control bone marrow (Figure 3.31).

Chapter 3. Results

CD19

CD3

CD8

CD3

CD4

0.53 %

0.99 %

0.75 %

0.86 % 0.46 %

0.74 % 13.8 %

14.2 % RAG2-/-γc-/- transplanted

with VAMP8-/- BM

14.2 % 52 %

RAG2-/-γc-/- transplanted with VAMP8+/- BM 0.07 % 58 %

B220

RAG2-/-γc-/-untransplanted C57BL/6

untransplanted

Figure 3.31. Bone marrow cells from VAMP8^-/^- sick mice develop into normal B-lymphocytes in RAG2 ^-/^- γc^-/^- mice: Dot plots showing the development of B and T lymphocytes in the blood from RAG2 -/^- γc^-/^- mice transplanted with bone marrow from VAMP8⁺/^- control and VAMP8^-/^- small and sick mice after 4 weeks. B lymphocytes developed within 4 weeks in the recipient of both types of bone marrow (Top panel). However, there are no CD4⁺CD3⁺ (bottom panel) or CD8⁺CD3⁺ (middle panel) T lymphocytes in the RAG2 ^-/^- γc^-/^- mice transplanted either with bone marrow from VAMP8^-/^- small and sick or VAMP8⁺/^- control mice. RAG2 ^-/^- γc^-/^- untransplanted control mice show no B- and T-lymphocytes.

*Transplantation was done at the central animal facility and irradiation of the RAG2 ^-/^- γc^-/^- mice was carried out at Strahlentherapie und Radionkologie by Dr. Mirko Nitsche and analysis was done with Dr.

Ralf Dressel at Department for Immunogenetics, Georg August Universität Göttingen.

Analysis of blood of all the RAG2 ^-/^- γc^-/^- mice transplanted with bone marrow from VAMP8⁺/^- control mice, showed CD4⁺CD3⁺ and CD8⁺CD3⁺ T lymphocytes by 6^th- 9^th week after transplantation.

In first experiment, there were few T lymphocytes in the RAG2 ^-/^- γc^-/^- recipient of VAMP8^-/^- bone marrow after 6 weeks, however, these mice developed CD4⁺CD3⁺ and CD8⁺CD3⁺ T lymphocytes after 9 weeks of transplantation in comparable numbers to the control mice (Figure 3.32). The RAG2 ^-/^- γc^-/^- recipient of VAMP8⁺/^- bone marrow developed CD4⁺CD3⁺ and CD8⁺CD3⁺ T lymphocytes after 6 weeks. After 8 weeks, the

Chapter 3. Results

RAG2 ^-/^- γc^-/^- mouse transplanted with control bone marrow died with symptoms of Graft-versus-Host Disease where ‘Graft’ was the donated bone marrow and the ‘Host’ was the recipient RAG2 ^-/^- γc^-/^- mouse. This indicates that this recipient mouse developed a functional immune system that eventually recognized the host as non-self and triggered a severe immune response leading to the death of the recipient.

CD3 VAMP8-/- small and sick bone marrow

recipient RAG2-/-γc-/- mouse

VAMP8+/- bone marrow recipient RAG2-/-γc-/- mouse

Figure 3.32. Bone marrow cells from VAMP8^-/^- sick mice develop into normal T-lymphocytes in RAG2 ^-/^- γc^-/ -mice: Dot plot data showing the development of T-lymphocytes in blood from RAG2 ^-/^- γc^-/^- recipients of bone there were almost no T lymphocytes in the VAMP8^-/^- bone marrow recipient at this stage (B). However, after 9 weeks, these mice developed both CD4⁺CD3⁺ and CD8⁺CD3⁺ T lymphocytes (C) in numbers comparable to the C57BL/6 control mice (D). ^*Transplantation was done at the central animal facility and irradiation of the RAG2 ^-/^- γc^-/^- mice was carried out at Strahlentherapie und Radionkologie by Dr. Mirko Nitsche and analysis was done with Dr. Ralf Dressel at Department for Immunogenetics, Georg August Universität Göttingen.

Chapter 3. Results

Figure 3.33. Bone marrow cells from VAMP8^-/^- sick mice develop into normal T-lymphocytes in RAG2 ^-/^- γc^-/ -mice: Blood analysis of transplanted RAG2 ^-/^- γc^-/^- mice to look for the development of T- and B- lymphocytes 6 weeks after transplantation. All the RAG2 ^-/^- γc^-/^- recipient mice transplanted with VAMP8⁺/^- or VAMP8^-/^- bone marrow developed CD4⁺CD3⁺ and CD8⁺CD3⁺ T lymphocytes and B220 bearing B-lymphocytes in similar numbers. ^*Transplantation was done at the central animal facility and irradiation of the RAG2 ^-/^- γc^-/^- mice was carried out at Strahlentherapie und Radionkologie by Dr. Mirko Nitsche and analysis was done with Dr. Ralf Dressel at Department for Immunogenetics, Georg August Universität Göttingen.

These experiments show that the T-lymphocytes progenitors of the VAMP8-/- mice do not have an inherent maturational defect. It is quite likely that the stromal cells of the thymus in VAMP8^-/^- small and sick mice are abnormal hence they fail to support the development and maturation of the thymocyte precursors.