Clasificación de linfomas en perros

1. Lymphoid Organs
Lymphocyte development and trafficking light
the way in lymphoma diagnosis
Peter F. Moore
pfmoore@ucdavis.edu

2. Immunophenotyping - cell lineage determina:on
Principles of integra:ve diagnos:cs for leukocy:c diseases
Lymphocyte development in primary lymphoid organs
Lymphoma in specific lymphoid domains
Lymphocyte an:gen receptor gene rearrangement - diagnosis of lymphoma
Outline:
Secondary lymphoid organs (lymph nodes and spleen) and lymphocyte trafficking
Plasma cell trafficking and mul:ple myeloma
Ter:ary lymphoid organs - lymphocyte trafficking to skin and gut
Ter:ary lymphoid organs - lymphoma in ter:ary lymphoid organs

3. Challenges in immunopathologic
inves>ga>on
CELL LINEAGE
Immunophenotyping
NATURE OF THE PROCESS
Molecular clonality

4. T lymphocytes: func>onal markers
CD3, CD4, CD5, CD8, CD11d, CD18, CD30, CD45, CD45RA
B lymphocytes: func>onal markers
CD5, CD18, CD19, CD20, CD21, CD79a, CD79b, CD45, CD45RA
Pax-5 (B cell Tf), Mum1/IRF4 (plasma cell Tf)
Granzyme B (cytotoxic T cells)
Bold = detectable in formalin fixed :ssues
His>ocytes: func>onal markers
CD1a, CD14, CD11b, CD11c, CD11d, CD18, CD80, CD86,
Iba-1, CD163, CD204, E-cadherin, Calprotec>n (L1)

5. IHC - formalin-fixed >ssue
CD3ε - mAb - CD3-12 (UCD, AbDSerotec) - many species
CD79a - mAb - HM57 (AbDSerotec) - many species
CD11d - mAb - CA18.3C6 (UCD) - dog only
CD18 - mAb - CA16.3C10 dog (UCD); FE3.9F2 cat (UCD); CA18.2G1 horse (UCD)
CD20 - polyclonal (LabVision) - many species
CD30 - mAb - BerH2 (Santa Cruz) - dog, horse
CD45 - mAb - CA12.10C12 (UCD) - dog only
CD45RA - mAb - CA21.4B3 (UCD) dog; mAb - B220 (BD/Pharmingen) - cat
CD90 (Thy-1) - mAb - CA9.GA11 (UCD) - dog only
MPO - mAb - CA25.7G1 (UCD) - dog, cat
UCD = Leukocyte An>gen Biology Laboratory (LABL) - pfmoore@ucdavis.edu

6. IHC - formalin-fixed >ssue
E-cadherin - mAb - clone 36 (BD/Pharmingen) - many species
c-KIT - rabbit polyclonal (Dako) - many species
Ki-67 - prolifera>on marker - mAb - MIB-1 (Dako; Santa Cruz) - many species
Granzyme B - cytotoxic granule protein - rabbit polyclonal (Spring Bioscience) - dog,
cat, horse
Calprotec>n (L1) - mAb - Mac387 (AbCAM) - dog, cat
Pax-5 - B cell Tf - (BD/Pharmingen)- dog, cat, horse
Mum1/IRF4 - plasma cell Tf - mAb - MUM1p (Dako) - dog, cat
CD163 - mAb - AM-3K; CD204 - mAb - SRA-E5 (Transgenic, Kobe, Japan) - dog, cat, horse
Ionized calcium binding adaptor-1 (Iba-1) - polyclonal - dog, cat, horse

7. Challenges in immunopathologic
inves>ga>on
CELL LINEAGE
Immunophenotyping
NATURE OF THE PROCESS
Molecular clonality

8. Lymphocyte an>gen receptor genes consist of many segments
(V)ariable, (D)iversity, and (J)oining genes undergo random
soma>c recombina>on to create the complete V exon - V(D)J
The adap>ve immune system u>lizes B and T lymphocytes to recognize and respond
to an almost limitless variety of an>gens (~1012) via their an>gen receptors
The genome (~2x104 genes) vs. Required diversity (~1012)
v v
c
c
SOLUTION: The Variable exon is formed
by recombina>on of mul>ple genes
An>gen binding occurs via the Variable Domains
of the an>gen receptor proteins Ag

9. An>gen receptor locus topology
Oligoclonality is not demonstrable!
C
C
C
C
C
V V V V V J J J J J
... IGH... (class switching)
TRD
V-(D)-J-C
1 rearrangement per chromosome
D
D
or
IGH
TRB
TRB
TRA
TRG
TRD
IGH
IGK
IGL
LOCI: All loci All loci
V-J-C1 V-J-Cn
...
TRGdog n=8 TRGcat n=6 TRGhorse n=17 Tandem V-J-C casseges
> 1 rearrangement per chromosome
TRG dog cat horse
V V J J Cn
V V J J C1
...

10. Lymphocyte an>gen receptor gene rearrangement
n *random bases - terminal transferase
RSS - recombina>on signal sequence
Splice donor site
Splice acceptor site
Junc%onal diversity* (1012)
(J)oining genes
(V)ariable genes
(D)iversity genes
V n J C (C)onstant gene
D n
Molecular fingerprint Random bases added (n) or deleted from the
junc:ons between V, D and J genes
Combinatorial diversity* (102 - 106)
PCR clonality Primers
CDR3 = complementarity determining region 3 or hypervariable region.
It is the major contributor to an:gen (Ag) binding.
CDR3

11. Lymphoid Organs
Primary Lymphoid Organs (central)
Thymus, Bone Marrow - lymphocyte development, an:gen receptor
gene rearrangement
Secondary Lymphoid Organs (peripheral)
Spleen, Lymph node - lymphocyte responses to an:gen
Ter>ary Lymphoid organs (peripheral)
Associated with mucosal sites (gut, lung) and skin

12. Thymus
panCK
C
M
Intrathoracic por>on - anterior medias>num
Cervical por>on - ruminants
M C
Cortex
Medulla
Hassall’s Corpuscle

13. Fagy encroachment
Thymus - age related involu>on
T cell pool
T cell input

14. T cell an>gen receptor genes TRDelta, TRGamma, TRBeta and TRAlpha rearrange
in a sequen>al manner
Rearrangement order:
TRG is func>onally rearranged in γδ T cells, and is present as a ves>gial rearrangement
in αβ T cells. TRD is deleted on at least one chromosome in αβ T cells.
TRD... TRB...
αβ T cells
TRA
TRG
γδ T cells
T cell development - thymus
TRD is deleted aher TRA rearranges in αβ T cells
TRG is an ideal molecular clonality target in ALL T cells

15. T cell development - thymus
TCRG/TCRD TCRA/TCRB
TCRαβ
TCRγδ
Ag binding
CD3
GENES:
Primary repertoire of TCRs - selected for self MHC restric:on (posi>ve
selec>on) and self tolerance (nega>ve selec>on) to yield the func>onal
repertoire
Gene rearrangement of germ line T cell an:gen receptor (TCR)
genes generates TCR diversity (primary repertoire)
The T cell an:gen receptors (TCR) on γδ T cells and αβ T cells associate with the
CD3 signaling complex, which is common to both receptor types.

16. Thymus - cell produc>on
αβ T cells
CD4+ T helper & T reg cells
MHC-II restricted
CD8+ cytotoxic T cells
MHC-I restricted
Peripheral (20) lymphoid organs
Major component of the Adap>ve
Immune System
γδ T cells
CD4-CD8- or CD8+
MHC unrestricted
Epithelia, splenic red pulp
Epithelial repair
Innate/adap>ve immunity -
bridge

17. THYMUS - αβ T cells
Naive T cells - exported from the thymus - recirculate
between blood and secondary lymphoid organs
Effector memory T cells - wider migratory range -
recirculate between blood and ter>ary lymphoid organs
(e.g. cutaneous or mucosal sites)
Central memory T cells - retain migratory path of
naive T cells

18. Bone marrow - B cell development
Bone marrow major site for B cell development
Immunoglobulin (Ig) gene rearrangement - produces surface Ig
an:gen receptor
IGH IGK IGL
+ or
Ig heavy chain
gene
κ light chain
gene
λ light chain
gene
mouse
human
dog cat horse...
human
Receptor diversity - bone marrow (most species)
and gut (birds, ruminants)
V gene soma>c hypermuta>on - affinity matura:on - occurs in
germinal centers of LN & spleen

19. B cell an>gen receptor gene rearrangement
B cell an>gen receptor complex - simplified. The integral membrane Ig molecule associates with
a signaling component - CD79. The V-D-J junc:on (CDR3) is the most important contributor to
an:gen binding in both the heavy chain and the light chain.
s-IgM / s-IgD
CD79 (Igα, Igβ)
Heavy chain IGH - VnDnJ C
Light chain IGL - VnJ C
IGK - VnJ C
Ag binding
C
C
V V
V
C
V
C

20. B cell development - Ig receptor diversity
COMBINATORIAL DIVERSITY
Large # V(D)J segments
Bone Marrow - most species
COMBINATORIAL DIVERSITY - LIMITED
Small # V(D)J segments
Diversity generated in gut - Birds, ruminants
MECHANISM
Gene conversion and/or soma:c hypermuta:on

21. B cell development - bursa
Bursa of Fabricius - chicken 1 wk
Bursal involu>on - chicken 12 wks

22. B cell development - Peyer’s patch
Goat 1 mth - Con:nuous ileal PP

23. B cell development - Peyer’s patch
Ileum: con:nuous PP 1-2 m
Jejunum: discrete PP 2-4 cm
Primary lymphoid organ Involutes 4 -6 months of age
Secondary lymphoid organ - no involu:on

24. Bone marrow - B cell produc:on
B cells are produced in the BM throughout life in
mammals
Mature naive B cells enter the blood and traffic to 2°
lymphoid organs - via HEV
B cells are agracted to (or form) follicles in 2° lymphoid
organs by specific chemokine interac:ons mediated by
follicular DC

25. Peripheral lymphoid organs
Lymphoid responses occur in peripheral lymphoid organs
2° lymphoid organs - lymph nodes, spleen, Peyer’s patch
3° lymphoid organs - mucosal sites and skin
Lymphocyte responses to an>gen occur efficiently in
these sites

26. Lymph node
Afferent lympha>cs - drain :ssue
Follicular domains (± germinal centers - GC) - B cells
Paracor>cal domains - T cells and dendri:c APC
Medullary sinuses and cords - short lived plasma cells
B cells, T cells recirculate from lymph to blood and enter
lymph nodes via high endothelial venules (HEV)
Recircula:ng lymphocytes use homing receptors which
recognize addressins on HEV

27. GC
Efferent lympha>c
Medullary sinuses Medullary cords
2o follicle
1o follicle
Paracortex
Afferent lympha>c
Subcapsular sinus
(T cell domain)
(B cell domains)
HEVs
GC

28. Dark zone (GC)
Light zone (GC)
Mantle zone
Marginal zone
(prolonged follicular hyperplasia)
Follicular architecture
GC = germinal center
FDC = follicular dendri:c cell
FDC network

29. CXCL13
CXCR5
Follicle forma>on
Follicular DCs
Follicular lymphocytes B cells
Follicular TH cells

30. Lymph node - follicular domain
GC Light zone
GC Dark zone
Mantle zone
“:ngible body” macrophages

31. Marginal zone hyperplasia - LN
Marginal Zone
GC - Dark zone
Mantle zone
GC - Light zone

32. Germinal Center Responses
Dark zone
Ig class switch
Mantle zone
Light zone
IGH-V muta>on
Ig affinity matura>on
(+)
IGH-V muta>on
(-)
Lymphoma
Target other genes
Soma>c hypermuta>on of IGH (and IGK, IGL) variable genes is a normal biological process in follicular germinal centers
of lymphoid :ssues. It has profound posi:ve implica:ons for the humoral immune response to an:gen. However, if
muta:on extends to other genes, lymphomas can eventuate. Many B cell lymphomas have a follicular/germinal center
origin, and B cell lymphomas are 3x more common than T cell lymphomas.
Lymphoid follicle

33. Diffuse large BCL
Centroblas>c
Immunoblas>c
T cell/his>ocyte rich
Anaplas>c
WHO classifica>on - Mature B cell neoplasia
Marginal zone BCL
Nodal
Splenic
MALT
Mantle cell BCL
Extramedullary Plasmacytoma
Follicular BCL
Mul>ple myeloma
B-CLL/Small lymphocy>c BCL
Burkig-like BCL
B
Indolent - ini>ally
Angiocentric BCL - “LYG”

34. Marginal Zone
GC - Dark zone
Mantle zone
MZL
MCL
DLBCL
GC - Light zone FL
MZH

35. GC
GC - centroblasts
Follicle - marginal zone B cells
LN - marginal zone lymphoma

36. B cell molecular clonality - lymphoma
PCR primers:
V n J C
D n
IGH
↓Sensi>vity IGH molecular clonality
IGH-V muta>on
Soma:c hypermuta:on is posi:vely selected for in CDR regions, but also targets framework regions (FR2 and FR3) of IGH (and
IGK, IGL) where PCR primers are posi:oned. If muta:on prevents primer annealing failure of PCR amplifica:on of CDR3 results.
The use of 2 FR primers (as indicated) is helpful.
2 3

37. Ig-κ locus is silenced in Ig-λ expressing B cells (90%) by KDE rearrangement
Ig Kappa locus - KDE
IGK
KDEv
V J KDE
C
KDEi KDE = Kappa dele:ng element
RSS
RSS = recombina:on signal sequence
RSS
RSS
Assessment of KDE rearrangements increases sensi>vity of B cell clonality determina>on
J
V KDE
n
IGK
KDEi PCR - dog, cat and horse
KDEv PCR - dog and cat
✂
Occurs during development in BM prior to germinal center - not mutated

38. 13-05-29 - Feline DSH, F, 9 yrs
1 month history of progressive upper respiratory obstruc:ve noises, and history of
chronic snoring. Retropharyngeal mass detected and biopsied by endoscopy
IGH2
polyclonal
IGH3
polyclonal
KDEi
clonal
DX: Retropharyngeal mass: large B cell lymphoma
Discussion: The cytological atypia of the large lymphoid cells and their B cell
immunophenotype suggests large BCL. Polyclonal IGH - somatic hypermutation of
the primer recognition sites or insufficient V gene coverage. Sufficient numbers of
residual reactive B cells contributed the polyclonal result. The KDEi rearrangement
(B cell kappa locus) was clonal thus confirming the diagnosis of lymphoma.
Pax5/CD3

39. Lymph node - paracor>cal domain
ParaCortex
HEV
HEV
F

40. Skin Dendri>c Cells - Langerhans cells, dermal DC
Ag driven migra>on
Lympha>c
Dermal DC
LC
SKIN
CCR7
CCL21
interdigita>ng DC in
paracor>cal domain Peripheral LN
LC and dermal DC express CCR7 to leave skin; they are awracted to the chemokine ligand CCL21 (red dots),
which is expressed on lympha:c endothelium and within the paracortex of lymph nodes

41. HEV
L
E
LN - trafficking
Naïve T cell
CCR7
αLβ2
L-selectin
PNAD CCL21
High endothelial venule
PNAD = peripheral lymph node addressin

42. Mycosis fungoides
Pagetoid re>culosis
Sézary syndrome
Peripheral TCL - “inflamed”
Peripheral TCL - “indolent”
Peripheral TCL - “unspecified”
SC “panniculi>s-like” TCL
Cutaneous TCL
WHO classifica>on - Mature T cell neoplasia
Extranodal - other
Hepatosplenic TCL
Hepatocytotropic TCL
Peripheral TCL - “unspecified”
Intravascular lymphoma
LGL leukemia
T-LGL CLL
T-LGL ALL
Nodal TCL
Peripheral TCL - “unspecified”
T-zone TCL
Anaplas>c large TCL
T
Indolent - ini>ally
Indolent - some forms
Intes>nal TCL Enteropathy associated TCL

43. PTCL - LN
CD3
Ki-67
Large T cells

44. F
paracortex
F
Intermediate T cells
HEV
TZL - LN
GC
paracortex
paracortex
Ki-67

45. Lymph node - medullary cords
MC
MS
MS
MC
Hematopoie>c environment
Short lived plasma cells

46. Spleen
Monitors blood borne an>gens
Splenectomy - reduced ability to handle bacteremia
White pulp (WP) - periarterial lymphoid sheath (PALS) (T
cells) and lymphoid follicles with op:onal germinal
centers (GC) (B cells)
Marginal zone (MZ) - normally present - enriched for
dendri:c cells, B cells
Red Pulp (RP) - red pulp cords enriched for macrophages
and γδ T cells; short lived plasma cells (B cell responses in
white pulp)

47. Canine spleen Follicle/GC
PALS Marginal zone/ellipsoids

48. Ellipsoids - CD18
Canine spleen Ellipsoids
Red pulp Ellipsoids - CD204

49. Spleen - lymphoid domains
TCRγδ
PALS
RP
GC
PALS
CD21
GC
MZ
GC
PALS
TCRαβ
GC
MZ
RP
CD11d
MZ

50. β2 integrin expression in splenic his>ocytes*
GC
Splenic Capsule
Central artery
CD11d
CD11c
Sinus
Red pulp cords
*β2 integrins (CD11/CD18) are heterodimeric leukocyte
adhesion proteins essential to leukocyte function
β
α
β2
αL
αM
αX
αD
CD18 CD11d
CD11b
CD11c
CD11a
Marginal zone
PALS (T cells)
Follicle (B cells)
Trabecular vein

51. CD11c
CD11d

52. GC
MZ
RP
CD11c
GC
MZ
RP
CD11d
Innate immune responses
Adaptive immune responses
β2 integrin expression in spleen*

53. CD11d
WP
MZ
RP
T-CLL - SGL type
Hepatosplenic T cell lymphoma (HS-TCL)
Hemophagocy>c HS (HHS)
Splenic red pulp - CD11d+ diseases

54. Liver - CD3
HS-TCL Liver Liver
Liver - CD11d
HS-TCL Spleen

55. Plasma cell trafficking
Medullary Sinus
Medullary Cord
Splenic red pulp
Plasma cells generated in 2° lymphoid organs (lymph node and
spleen) - short- and long-lived
Short-lived plasma cells (~ 4 - 6 weeks)
Home to medullary cords of lymph nodes or splenic red pulp
Long-lived plasma cells (years)
Home to bone marrow

56. Mul>ple Myeloma
Origin: Bone marrow homing plasma cell
Mul:focal plasma cell neoplasm of long lived plasma cells
Clinical features (variable expression):
Monoclonal serum Ig
Bone destruc>on (pathological fractures)
Hypercalcemia
Anemia
Amyloidosis (AL)
Wide spectrum of behavior from indolent to aggressive with
dissemina:on to soh :ssues

58. Peripheral Lymphoid Organs
Ter>ary Lymphoid organs
Skin associated lymphoid >ssue (SALT)
Lymphoid >ssue associated with mucosal sites -
gastrointes:nal and respiratory tracts - (GALT BALT NALT)

59. Lymphocyte Trafficking
and
Tissue localiza>on of disease in ter>ary
lymphoid >ssues

60. The Biology of Skin Lymphocytes
Effector memory T cells
Regulatory T cells - Treg
B cells rarely encountered in normal skin
Lymphocyte recruitment to skin
DC migra:on and an:gen presenta:on
Dendri>c cell imprin>ng
Resident and Migratory T cells

61. Skin Dendri>c Cells - Langerhans cells, dermal DC
Ag driven migra>on
Lympha>c
Dermal DC
LC
SKIN
CCR7
CCL21
interdigita>ng DC
Peripheral LN
LC and dermal DC express CCR7 to leave skin; they are awracted to the chemokine ligand CCL21 (red dots),
which is expressed on lympha:c endothelium and within the paracortex of lymph nodes

62. LN Paracortex
Home to SKIN
Effector/Memory T cell
CCR4
αLβ2
E-selec>n CCL17
Dermal endothelium
CLA
Fucosyl Tr VII
Naive T cell - traffics to LN
Dendri>c Cell - an>gen presenta>on
MHC-pep>de
TCRαβ
B7
CD28
CCR = chemokine receptor CCL = chemokine ligand CLA = cutaneous lymphocyte an:gen
L-selec>n
αLβ2
CCR7
1 2

63. Lymphocyte recruitment - to skin
Chemokine ligands (and cytokines) are produced by perivascular Mast cells and Dermal DCs, and Kera:nocytes and LCs. In
inflamma:on - increased produc:on of chemokines and inflammatory cytokines - vastly increases trans-endothelial cell traffic.
Addressin
Chemokine ligand Adhesion ligand
Dermal vascular
endothelium
Mast cells
Dermal DC
LC
KC
Chemokine ligands - Cytokines upregulate
E-selec:n CCL17
Effector/Memory T cell
αLβ2 Adhesion molecule
CCR4
CD11a
Chemokine receptor
Homing molecule
CLA
1 2 3
ICAM-1,2
Roll

64. Human skin: Human resident memory T cells (TRM) exist as 2 main popula:ons, which largely differ with respect to CD103
expression; both subsets largely express CD69 (an ac:va:on marker). Neither subpopula:on leaves the skin. There are also 2 main
popula:ons of recircula:ng skin homing T cells (CLA+): T central memory cells (TCM), which express CCR7 and L-selec:n and home
to all lymph nodes, and T migratory memory T cells (TMM), which express only CCR7 and migrate to skin draining lymph nodes via
lympha:cs. Watanabe, Science Transla:onal Med, (2015), 7(279):279ra39
Normal human skin - effector/memory T cells dominate
CD69+ CD103+
CLA+ TRM
CLA+ TRM
CD69+ CD103-
CLA+ TCM
CCR7+ L-selec>n+
CCR7+ L-selec>n-
CLA+ TMM

65. TRM cells -
Generated in local skin immune responses - globally protect skin.
Provide rapid local immune protec:on - infec:on/an:gen exposure.
Inflamma>on - Atopic derma::s, psoriasis
Malignancy - Mycosis fungoides - Epitheliotropic cutaneous T cell
lymphoma (eCTCL)
TCM cells -
Generated in local skin immune responses.
Recirculate between blood, lymph nodes and skin - far less effec:ve
in local skin immune protec:on
Malignancy - Sezary syndrome (leukemic CTCL)
Clark RA. Sci Transl Med. 2015; 7(269): 269rv1.

66. Mycosis fungoides
Pagetoid re>culosis
Sézary syndrome
Peripheral TCL - “inflamed”
Peripheral TCL - “indolent”
Peripheral TCL - “unspecified”
SC “panniculi>s-like” TCL
Cutaneous TCL
WHO classifica>on - Mature T cell neoplasia
Extranodal - other
Hepatosplenic TCL
Hepatocytotropic TCL
Peripheral TCL - “unspecified”
Intravascular lymphoma
LGL leukemia
T-LGL CLL
T-LGL ALL
Nodal TCL
Peripheral TCL - “unspecified”
T-zone TCL
Anaplas>c large TCL
T
Indolent - ini>ally
Indolent - some forms
Intes>nal TCL Enteropathy associated TCL

67. Skin homing T cell lymphoma

68. Classical MF
TCR usage: TCR αβ ≈ TCR γδ

69. Pagetoid MF TCR usage: TCR γδ exclusively
TCR γδ
TCR αβ

70. Canine MF - topography
Mucocutaneous junc>ons/oral >ssues (~ 60% dogs)
Footpads/interdigital fold (~ 15% dogs)
Extends the range of skin trafficking T cells to
include >ssues of the oral cavity
Moore et al. Vet Derm. 2009, 20:569-576

71. Canine MF - topography
Facial mucocutaneous junc>ons and feet - common sites -
canine atopic derma>>s
These sites were involved in ~ 50% dogs with MF
Atopic derma>>s - risk factor for MF in dogs
γδ T cells expanded in the epidermis of dogs with atopic
derma>>s
Emergence of MF from a chronic inflammatory background
highly likely
Olivry et al. The Am J of Dermatopath 1997, 19:477-486
Santoro et al. Vet Derm 2007, 18:101-106

72. Polyclonal background par:cularly high in inflamed
cutaneous T cell lymphoma
Inflamed non-epitheliotropic T cell lymphoma ...
Reac:ve T cells are αβ T cells - most will have TRD
deleted
If the lymphoma cells are γδ T cells (~50% incidence)
they will have clonal TRD

73. 17-01-60 - Canine, Dachshund, FS, 10 yrs
7 yr history of pedal and vulvar pruritus; swelling of digits (leh pelvic limb) leading to
amputa:on (1 yr ago). Biopsy 1 DX: eosinophilic granulomatous pododerma::s.
Biopsy 2 DX: pyogranulomatous to pleocellular pododerma::s. Dog then developed
mul:focal nodules on lips and digits. Biopsy 3 DX: leukocy:c round cell malignancy.
P3
P2

74. CD3 CD3 GrB GrB

75. DX. Digit: inflamed large granular (LGL) T cell lymphoma with par:al CD3 loss
TRG1 TRG2 TRG2rep TRD
The clonal TRD result indicates the lymphoma is likely a prolifera:on of γδ T cells. This is
rela:vely common in cutaneous lymphomas of dogs.
Discussion:
The inflammatory T cell component (αβ T cells) hindered recogni:on of the clonal TRG peak.
Molecular clonality: Clonal TRG2 and TRD
Immunophenotype: CD3± GrB+
17-01-60 - Canine, Dachshund, FS, 10 yrs

76. Gut associated lymphoid >ssue
Intes>nal immune system
Diffuse mucosal associated lymphoid :ssue (MALT) -
only an:gen experienced lymphocytes (effector/
memory cells) can traffic here
Organized MALT - Peyer’s patches and mesenteric LN
- 2° lymphoid organs - naive lymphocytes can traffic
here

77. From: Nature Reviews Immunology (2004) 4, 290
Small intes>nal immune system
3° 2°
3° 2°
Lympha:cs
Lympha:cs

78. Peyer’s patch - aggregated lymphoid follicles
Jejunum: discrete PP 2-4 cm

79. Organized MALT
Peyer’s patch
Dome area (M cells)
Lymphoid follicle
(B cells)
Interfollicular area
(T cells)

80. Diffuse MALT
Jejunum
plasma cells
lymphocytes
pIg-R
(IgA)
LPC
IEC
villus
crypt
IEL
LPL
IgA
pIg-R = poly-Ig receptor for IgA
LPC = lamina proprial compartment
LPL = lamina proprial lymphocyte
IEC = intra-epithelial compartment
IEL = intra-epithelial lymphocyte

81. Diffuse MALT (LPL and IEL) - largely a T cell environment - few B cells
IELs exhibit dis>nc>ve phenotypic subsets - differ markedly from PBL
CD8αα T cells predominate - unique role in mucosal immune
surveillance
20 - 40% of IELs are small granulated T cells (express GrB)
Feline IEL express the mucosal integrin - CD103 (αEβ7)
Plasma cells - secrete dimeric IgA - cluster in the crypt lamina propria
Feline small intes>ne: diffuse MALT

82. IgA
Ag
IgA response
Effector
Func>on
Induc>on

83. Lymphocyte recruitment to gut
How are the migratory pathways of naive lymphocytes
redirected to gut?
Dendri>c Cell Imprin>ng

84. DC
Peyer’s patch
interdigita>ng DC
Mesenteric lymph node
(MLN)
DC imprint effector/memory T cells
for mucosal homing
β7 integrin expression

85. Naïve T cell
MLN Paracortex/Peyer’s patch
Dendri>c Cell
Re>noic acid
αEβ7
Effector/Memory T cell
CCR9
α4β7
Home to GUT
CCL25
MAdCAM
Mucosal endothelium
MHC-pep>de
TCRαβ
B7
CD28
MAdCAM = mucosal addressin cell adhesion molecule

86. plasma cells
lymphocytes
pIg-R
(IgA)
LPC
IEC
villus
crypt
IEL
LPL
CCL25
MAdCAM
Mucosal endothelium
CCR9
α4β7
T mem
CCL25
LPL
1 10 100 1000 10000
0
200
400
600
800
1000
flow979.020
FL1-H
SSC-H
αEβ7
70%
1 10 100 1000 10000
0
200
400
600
800
1000
flow980.029
FL1-H
SSC-H
CD3
91%

87. Mucosal homing T cell lymphoma

88. Inflammatory bowel disease (IBD) - cat
Polyclonal (reac>ve)
Size (bp)
Size
(bp)

89. Mucosal epitheliotropic small T cell lymphoma - cat
Clonal (lymphoma)
Size (bp)
Size
(bp)

90. Transmural T cell lymphoma - cat

91. Localized to the diffuse MALT - due to expression of
mucosal homing receptors
Mesenteric lymphadenopathy may develop with
local disease progression
Metastasis beyond the gut and draining lymph node
is a late event
Intes>nal T cell Lymphoma

92. Integra>ve diagnos>cs - Leukocy>c diseases
Clinical and clinico-pathological data
Morphological data - histology/cytology
Immunophenotyping - :ssue; cytology; flow cytometry -
preferably with reagent panels
Molecular assessments for lymphoma - lymphocyte
an:gen receptor gene rearrangements
Disease outcome - did it all “add up”?