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T NK Cell
T NK Cell
T NK Cell
By:
Ahmed Makboul Ahmed
M.B.B.Ch, M.Sc
Assistant Lecturer, Clinical Pathology Department, South Egypt Cancer Institute
INTRODUCTION
LYMPHOCYTE MATURATION
T-CELL DIFFERENTIATION
There are two classes of T-cells: alpha beta T-cells and gamma delta T-cells. This distinction is
based on the structure of the T-cell receptor.
Alpha beta (αβ) T-cells
• Represent the majority of T-cells.
• They express CD4 or CD8 as well as CD5.
Gamma delta (γδ) T-cells
• <5% of all normal T-cells.
• Distribution: Found mainly in the splenic red pulp, intestinal epithelium, and other epithelial sites.
• Function: γδ T-cells serve as a first line of defense against bacterial peptides, such as heat shock
proteins.
o Involved in responses to mycobacterial infections and in mucosal immunity.
• Immunophenotype:
o Bright CD3, CD2+, CD7+
o CD4-, CD8- (a subset may be CD8+)
o CD5 dimmer positive / small subset may be negative
o CD16 subset+, CD56 subset+, CD57 subset+, CD94+
o They are smaller on forward scatter.
Immunophenotyping of normal γδ T-cells (pink population)
CD4+ T-cell subsets
1. T-helper
2. T follicular helper (TFH) cells
Function
• TFH cells provide help to B-cells in the context of the
germinal center reaction.
Immunophenotype
• They express the germinal center markers: BCL6 and
CD10.
oThese markers are also normally found on
germinal center B-cells.
• CD4+, CD57+ and CD279/PD1+, ICOS+
• They produce the chemokine CXCL13 and express
chemokine receptor CXCR5.
oIncreased expression of CXCL13 is seen in
angioimmunoblastic T-cell lymphoma (AITL).
3. Regulatory T-cells (T-reg)
• Regulatory T-cells (T-regs) are a subset of
CD4+ T cells whose function is to suppress
immune responses and maintain self-
tolerance
• Immunophenotype
osCD3 (−), CD5 (−)
oCD2 (+), CD7 (+)
oCD8 partial (+)
oCD56 (+), CD16 (+), CD94 (+), and CD57 small subset
oNK cells express various KIR antigens (CD158a,
CD158b, CD158e)
NK-cell immunophenotyping (Brown population)
Function:
Mechanism of killing:
Manifestations
• Most patients with T-PLL present with an elevated WBC. WBC is often markedly
increased, > 100 x 109/L.
oNot uncommonly, some patients may present with a WBC around 20–30 x 109/L that
often progressively increases in the course of disease.
• Hepatosplenomegaly (50–70%), generalized lymphadenopathy (50%), anemia and
thrombocytopenia, skin infiltration (20–25%), and serous effusions (i.e., pleural) (15%)
occur at various frequencies in patients with T-PLL.
MICROSCOPIC PATHOLOGY
Lymphocyte morphology
• Size
oSmall to medium sized.
• Nucleus
oContour: Round nuclear contours.
oChromatin: Moderately condensed nuclear
chromatin.
oNucleolus: Prominent nucleoli.
• Cytoplasm
oModerate amounts of pale blue, agranular
cytoplasm.
oCytoplasmic blebs are common.
Bone Marrow examination
• BM is almost always involved
oPattern of infiltration:
§ Interstitial pattern.
§ diffuse leukemic infiltrative pattern.
Morphologic variants
1. Small cell variant
• 20% of cases.
• Small neoplastic cells with inconspicuous or absent
nucleolus.
2. Cerebriform variant
• 5% of cases.
• Cells have irregular nuclear outline resembling
cerebriform nucleus of Sézary cells seen in MS/SS.
ANCILLARY STUDIES
1. Flow cytometric immunophenotyping
• Pan-T-cell antigens:
o CD7 and CD5 usually strongly (+).
o Positive CD26 in all cases.
o Cytoplasmic CD3 always (+)
o Surface CD3(+/-) and can be dim.
o TCR-αβ can be variably (+).
• Frequency of CD4 and CD8 expression:
o CD4(+), CD8(-): 60%
o CD4(+), CD8(+): 35%
o CD4(-), CD8(+): 4%
o CD4(-), CD8(-): 1%
• CD52 strongly positive in virtually all cases.
o Needed for treatment purposes (Anti-CD52).
• CD45 expression is bright unlike lymphoblasts.
o It can be negative in 5 – 10% of cases.
• Immature markers (CD34, TdT, CD1a) negative.
2. Immunohistochemistry
TCL1
3. Cytogenetic studies
• Chromosomal abnormalities are common,
observed in 60–70%, and the majority has a
complex karyotype.
Manifestations
• Multiple clinical forms:
oSmoldering and chronic.
oLymphomatous.
oAcute (leukemic).
• Cytoplasm
oMedium or scanty.
oBasophilic, agranular.
Bone Marrow examination
• Bone marrow infiltrates are usually patchy and subtle,
even in patients with a leukemic presentation, ranging
from sparse to moderate.
• Osteoclastic activity may be prominent, even in the
absence of BM infiltration by neoplastic cells.
Manifestations
• Skin involvement predominates in both MF and SS.
• Diverse skin lesions in MF.
• Erythroderma in SS; rarely pruritus without erythroderma.
• Onset of erythroderma more abrupt in SS than slowly evolving skin lesions in MF.
• Lymphadenopathy at presentation in SS, often develops during MF disease course.
MICROSCOPIC PATHOLOGY
Sézary cells morphology
Sézary cells exhibit distinctive cytologic features:
• Size
oMedium to large overall size.
oIn some patients with SS, the circulating
neoplastic cells are small in size. These
small circulating Sezary cells can be
mistaken for normal lymphocytes. However,
on careful review, the nuclear atypia is
apparent.
• Nucleus
oContour: Distinctly cerebriform nuclear
configuration.
oChromatin: Nuclear chromatin typically highly
condensed.
Bone Marrow examination
• Bone marrow is often not involved or only
minimal involvement.
CD3
MF/SS Prototypic Features
CD4 positive T-cell
neoplasms
- ALK+ ALCL comprises 3% of NHL in adults and 20–30% in children and adolescents.
Manifestations
• B symptoms, common.
• High-stage (III-IV) disease in majority of cases.
oPeripheral and intraabdominal lymphadenopathy.
oExtranodal involvement:
§ Skin, bone, soft tissue, and bone marrow.
§ PB involvement in subset of small cell variant of ALCL, ALK (+).
MICROSCOPIC PATHOLOGY
Morphologic variants
1. Classic (70%):
• Predominance of large cells with irregular nuclei and frequent large hallmark cells
2. Lymphohistiocytic (5–10%):
• Neoplastic cells admixed with abundant reactive histiocytes.
• In BM, the infiltrate is interstitial, which may be difficult to see due to small cell size
and a subtle infiltrate.
ANCILLARY STUDIES
1. Flow cytometric immunophenotyping
• Be careful with gating strategy!
• CD3 often negative.
• CD2, CD5, and CD4 expressed in 70% of
cases.
• CD30 positive.
• Side scatter often high
• Can be CD45 negative!!
2. Immunohistochemistry
• Expression of 1 or more T-cell-associated
antigens
oCD2, CD5, and CD4 expressed in 70% of cases.
oCD3 is negative in vast majority of the cases.
oCytotoxic-associated markers, TIA-1, granzyme
B, &/or perforin, expressed in most cases. CD30
• Pathogenesis
oSTAT3 mutations in > 40%; somatic STAT5B mutations in 2%
§ Provide prosurvival and growth signals.
oChronic antigenic stimulation resulting in proliferation of T-
cell LGLs
oInhibition of apoptosis resulting in accumulation of T-LGLs.
T-cell Large Granular Lymphocytes NK- Large Granular Lymphocytes
(T-LGL) (NK-LGL)
Functions Functions
1. Regulation of hematopoiesis. 1. Regulation of hematopoiesis.
2. Regulation of B-lymphocytes. 2. Key role in innate immunity.
3. Low NK-cell activity (MHC non-restricted 3. High NK-cell activity (MHC non-restricted
cell lysis and destruction of normal tissue) cell lysis).
4. Variable ADCC. 4. ADCC.
5. Contrasuppressor activity: Inhibition of 5. Immunosurveillance for spontaneously
other T-suppressor cells from inhibiting T- occurring neoplasms.
helper cell activity. 6. Resistance to viral infections.
Immunophenotype Immunophenotype
• CD3 +, CD8 +, CD4 −, CD57 +, CD56 small • sCD3 −, CD2 +, CD5 −, CD7 +, CD8
subset, CD16 −/very dim +, CD94 dim +, subset+, CD16 +, CD56 +, CD57 small
TCR-αβ + subset +, CD38 bright + and CD94 +.
CLINICAL FEATURES
Age: Median age at diagnosis of T-cell LGL leukemia is 60 years.
o 20-25% of patients are younger than 50 years.
Gender: T-cell LGL leukemia affects men and women equally.
Manifestations
• Most of the patients with T-cell LGL leukemia come to medical attention with cytopenia(s), frequently isolated
neutropenia.
o Pure red cell aplasia (PRCA) has been reported in 20 % of patients, and aplastic anemia has also been
reported.
o Thrombocytopenia occurs in about 20% of patients and ITP is seen at an increased frequency in patients
with T-cell LGL leukemia.
• Splenomegaly is observed in 25–50% of cases, whereas hepatomegaly and lymphadenopathy are very rare.
• Rheumatoid arthritis appears to be the most frequent autoimmune disease associated with T-cell LGL leukemia,
reported in up to 35% of patients.
o Serologic abnormalities (rheumatoid factor, antinuclear antibody, and polyclonal hypergammaglobulinemia)
are frequent, even in patients without clinical manifestation of an autoimmune disease.
MICROSCOPIC PATHOLOGY
LGL lymphocytosis
• Absolute LGLs range between 2 and 20 x 10⁹/L
(according to WHO guideline).
• Occasional cases LGLs range between 0.5 and 10 x
10⁹/L.
LGL morphology
Size: Small to medium in size.
Nucleus:
oContour: Round/slightly indented contour.
oNucleolus: Inconspicuous nucleoli.
oChromatin: Condensed chromatin.
Cytoplasm: Abundant pale cytoplasm, and fine
azurophilic granules.
Bone Marrow examination
• Presence of BM infiltrate is in favor of T-cell LGL leukemia.
• Interstitial/intrasinusoidal patterns common.
oMorphologically occult and difficult to identify.
oImmunostains helpful to identify T-LGL infiltrate.
ANCILLARY STUDIES
1. Flow cytometric immunophenotyping
• Common type of T-cell LGL leukemia (present in 70% of cases)
oCD3 (+) with coexpression of CD16 (+) and CD57 (+), CD4 (-), T-cell receptor αβ (+).
oT-cell antigen aberrancy (diminished CD2, CD5 or CD7 expression common).
oCD56 is generally negative in T-cell LGL leukemia, unlike normal/reactive T-LGL
cells.
§ However, uniform expression of CD56 can be seen in some T-cell LGL leukemia.
üCD56+ T-cell LGL leukemia is a clinically aggressive variant T-LGL leukemia.
However, these reported CD56+ cases are all CD57-negative.
oSurrogates of clonality
§ Uniform expression of a single KIR antigen: CD158a, CD158b, CD158e.
§ Uniform expression of T-cell receptor Vβ region.
T-cell LGL leukemia (Common type)
• T-cell LGL leukemia, TCR-γδ variant
oComprises 10% (5 – 15%) of all T-cell LGL leukemia.
oCD3 bright (+), CD8 (−), CD4 (−), CD57 (+) and TCR γδ (+).
oCD56 is often negative but can be partial/subset+ or positive together with
CD57.
§ In some cases, the leukemic cells may be negative for both CD56 and
CD57.
oCD5 is dimmer (+) or partial (+) and only occasionally completely negative.
oThe critical differential diagnosis is hepatosplenic γδ T-cell lymphoma.
Granzyme B TIA1
3. Genetic studies
• Identification of clonal TCR gene rearrangement by PCR helps for a diagnosis of T-cell
LGL leukemia.
• Presentation
oPatients with reactive LGL lymphocytosis often have cytopenia(s) and various clinical
symptoms related to underlying medical conditions, and, the expansion of LGL cells may
last for a prolonged period of time.
• Morphology
oTypical LGL morphology.
oLGL lymphocytosis shows no BM involvement; whereas T-
cell LGL leukemia almost always shows BM infiltration.
oTherefore, a BM biopsy with IHC panel is recommended to
confirm diagnosis of LGL leukemia.
• Immunophenotype
oNormal T-cell LGL immunophenotype (no aberrancy).
• Molecular studies
oPolyclonal/oligoclonal T-cell receptor γ.
Diagnostic criteria of T-cell LGL leukemia*
Major criteria
1. Flow-cytometric immunophenotyping revealing > 50% of the total PB or BM surface CD3-positive T-
cells to have two or more of the following:
o CD8 positive (may be dim).
o Uniform expression of CD16 or CD57 (>75% of cells positive).
o Loss of CD5 expression (partial or complete).
o Uniform expression of one or more of the KIRs CD158a, CD158b, and CD158e.
2. Intrasinusoidal BM or splenic infiltration by cytotoxic lymphocytes positive for one CD8 and one or
more of the cytotoxic markers TIA-1, granzyme B, granzyme M, or perforin.
Diagnosis of T-cell LGL leukemia requires three or more major criteria are present or two
major criteria and two or more minor criteria.
Manifestations
• Patients often present with fever, fatigue, weight loss, and abdominal discomfort due to
hepatosplenomegaly and, sometimes, with jaundice because of liver involvement.
• Lymphadenopathy is usually absent.
• PB often shows pancytopenia or bicytopenia(s) (anemia and thrombocytopenia).
o Cytopenia(s) may be a combination of hypersplenism, BM infiltrate, or abnormal cytokine release
by tumor T-cells with or without an underlying hemophagocytosis.
• Patients usually do not have peripheral lymphocytosis at initial presentation; however, a small
population of circulating neoplastic lymphocytes may be seen at the presentation in 50%.
MICROSCOPIC PATHOLOGY
Lymphocyte morphology
Size:
oIntermediate to large (blastic-like) cells.
Nucleus:
oContour: Irregular contour.
oNucleolus: Inconspicuous nucleoli.
oChromatin: Condensed but dispersed chromatin.
Cytoplasm:
oModerate amount of basophilic agranular cytoplasm.
oLymphoma cells may contain variable numbers of
azurophilic granules and, when abundant, may be
mistaken as LGL cells
• Although patients often do not have PB lymphocytosis, the
neoplastic cells are often present in peripheral blood in
variable numbers
Bone Marrow examination
BM is almost always involved in HSTL.
Pattern of BM infiltration
• The infiltrate is intrasinusoidal, and the infiltrate can be very subtle at the time of
diagnosis.
oOne of the important features of HSTL is that the lymphoma cells not only involve
sinuses but also expand the sinuses. This expansion of sinuses differs from T-
cell LGL leukemia with a sinusoidal involvement, which often shows a linear
single layer of cells.
oIn many cases, the infiltrate may be missed if IHC or flow cytometry study is not
performed.
oThe cells in the intrasinusoidal spaces are often monomorphic and medium-sized,
with slightly irregular nuclei, condensed chromatin, indistinct nucleoli, and
moderate amount of clear cytoplasm.
• As the disease progresses, the neoplastic infiltrate can go beyond the sinuses,
showing an interstitial or diffuse pattern.
a b c
(a & b) Bone marrow biopsy shows a lymphoid infiltrate. (c) CD3 highlights the infiltrate with a sinusoidal expansile pattern,
only very few tumor cells are scattered in the interstitial area.
Other findings in BM
• Dysplastic features involving 1–3 hematopoietic cell lineages can be frequently
observed in patients with HSTL.
oThis is likely a result of cytokine effects or due to the perturbation of the BM
microenvironment by lymphoma cells.
• Gene mutations
oSETD2 (a tumor suppressor gene) mutations
in about 70% of patients with HSTL.
oSTAT5B is detected in around 30%
oSTAT3 is less common (9%).
Subtelomeric probe for 7p (green) and 7q (red)
Features LGL HSTL
Age 60 (12–87) 34 (16–58) (Younger age than T-LGL leukemia)
Male/female 1:1 (Equal) 5:1 (Male predominance)
C/P o Underlying autoimmune disease or autoantibodies. o B-symptoms: Fever, weight loss, night sweats, and fatigue.
o Chronic infection. o Hemophagocytosis is common.
o Association with immunosuppression.
BM infiltrate o Interstitial and or intrasinusoidal linear pattern. o Intrasinusoidal/intravascular, with expansion of the sinuses.
o Reactive lymphoid aggregates common.
Immunophenotype CD5 dim (+), CD56 −/ or variably+, CD57 (+), CD5 often negative, CD56 (+), CD57 (−), CD16/CD94 frequently
CD16/CD94 dim bright (+)
Cytotoxic granules o TIA1 (+). o TIA1 (+).
o Granzyme B may be lost in about 30–40% cases o Granzyme B often negative
Cytogenetics/mutation o Mostly normal o i7q, +8
o STAT3 in 30-40%, and rare STAT5B. o SETD2, STAT5B, rare STAT3.
Clinical course Indolent Aggresive
Diagnostic criteria of HSTL*
Criteria supporting HSTL
1. B symptoms.
2. Massive splenomegaly.
3. Lymphoma cells expand BM sinuses.
4. Immunophenotype displays CD3 (+), CD5 (-), CD4 (-)/CD8 (-), CD56 (+), granzyme-B (-) and
TCR-γδ (+).
5. Isochromosome 7q or trisomy 8.
6. Monoclonal TCR gene rearrangement.
* Yabe M et al: Distinguishing between hepatosplenic T-cell lymphoma and γδ T-cell large granular lymphocytic leukemia: a
clinicopathologic, immunophenotypic, and molecular analysis. Am J Surg Pathol. 2017; 41: 82-93.
Criteria not supporting HSTL
1. Absence of splenomegaly.
2. Lymphadenopathy.
3. Extranodal site of involvement.
4. PB lymphocytosis > 5x109/L.
5. Neoplastic lymphocytes with azurophilic granules.
6. Evidence of infection by EBV, HIV or HTLV-1.
7. Immuophenotype with expression of CD5, CD8, CD57, granzyme B and TCR- αβ.
8. Negativity for monoclonal TCR gene rearragement
* Yabe M et al: Distinguishing between hepatosplenic T-cell lymphoma and γδ T-cell large granular lymphocytic leukemia: a
clinicopathologic, immunophenotypic, and molecular analysis. Am J Surg Pathol. 2017; 41: 82-93.
NK-cell Lymphoproliferative Neoplasms
NK-cell lymphoproliferative
neoplasms
Pathogenesis
• Overlap With T-Cell LGL leukemia
o Chronic persistent antigenic stimulation likely pathogenic
for both T-LGL leukemia and CLPD-NK.
o Dysfunctional activation of survival pathways and the
evasion of apoptosis.
o Activating STAT3 mutations detected in 30% of cases.
CLINICAL FEATURES
Age: Affects adults predominantly (median age: 60 years).
Manifestations
• CLPD-NK has a similar indolent clinical presentation as T-cell LGL leukemia.
• The number of circulating PB NK-cells usually remains stable for a long period of time, and
some cases have even been reported to show spontaneous regression.
MICROSCOPIC PATHOLOGY
Lymphocyte morphology
• Mature NK cells ≥ 2 x 10⁹/L in PB.
Nucleus:
o Contour: Round/slightly indented contour.
o Nucleolus: Inconspicuous nucleoli.
o Chromatin: Condensed chromatin.
• KIR antigens (CD158a, CD158b, and CD158e) can be assessed by flow cytometry to
provide information of NK-cell clonality.
• Molecular methods
oA clonal NK cell proliferation does not show TCR gene rearrangements.
oThe methods for NK-cell clonality assessment include:
§ Assessment of the pattern of inactivation of the X-chromosome (e.g., the human
androgen receptor assay (HUMARA).
§ Presence of STAT3 mutations (approximately 30–40% cases).
Diagnostic criteria of CLPD-NK
Habibe et al. “Chronic lymphoproliferative disorder of NK-cells: A single-institution review with emphasis on relative utility
of multimodality diagnostic tools.” European Journal of Hematology 100 (2018): 444–454.
A suggested algorithm in the workup of LGL proliferation
Pathogenesis
• EBV Positivity
oSupports viral oncogenic role.
oMay evolve from chronic active EBV infection.
CLINICAL FEATURES
• Age: Predominates in young to middle-aged adults.
• Manifestations
oGenerally acute onset of symptomatology.
oPatients usually are extremely ill, with fever, acute coagulopathy, hepatosplenomegaly,
pancytopenia, and abnormal liver function.
oIncreased LDH and increased ALT & AST.
oSome patients have a high WBC count due to the presence of circulating tumor cells.
oThere is frequent CNS involvement.
oThe disease course is fulminant; with multiorgan failure and disseminated intravascular
coagulation, death usually occurs within a few weeks.
MICROSCOPIC PATHOLOGY
Lymphocyte morphology
• ANKL cells could be quite variable in size and appearance.
oIn some patients the leukemic cells are pleomorphic,
larger with more open chromatin and distinct nucleoli.
oSome patients have blast-like morphology with
monotonous, medium-sized lymphocytes that have fine
chromatin and coarse azurophilic granules.
oIn other patients, the cells have been described similar
to LGL cells that the cytoplasm often contains variable
numbers of azurophilic granules
EBER
3. Genetic studies